Surface waters are contaminated by surface runoff, inland drainage and sewage discharge. Thus, aquatic vertebrates, like amphibians and fish, are main targets of a vast number of exogenous steroids or steroid-like chemicals and the number of reports about androgenic and estrogenic EDCs affecting development and physiology of amphibians. Most of these publications, however, focus on exposure to EDCs with just one single MOA, while studies examining the effects of simultaneous exposure to EDCs with different MOAs are rare, although they would reflect more real, natural exposure situations. Thus, such combination effects need to be evaluated more closely. The South African clawed frog was shown to be an appropriate model for the assessment of androgenic and estrogenic EDCs. Especially the male calling behavior of this species was shown to serve as highly sensitive and non-invasive biomarker, detecting very low, environmentally relevant concentrations of androgenic and estrogenic EDCs. Hence, the aim of the present study was to assess the effects of antiestrogenic EDCs on the male calling behavior of X. laevis and further evaluate the combined effects of EDCs with estrogenic and antiestrogenic MOAs on this endpoint to obtain information whether simultaneous exposure to EDCs with opposing MOAs can lead to an obliteration of some EDC effects, or whether the combined exposure substances can act synergistically and result in further impacts. To achieve this aim, the contraceptive 17a-ethinylestradiol was used as estrogenic EDC, while the pharmaceuticals tamoxifen and fulvestrant served as antiestrogenic model substances in this study. TAM and ICI are pharmaceuticals used to treat advanced breast cancer. By selectively modulating ER, TAM can exhibit distinct MOAs in different tissues. It inhibits transcriptional activity of ER in breast tissue and exhibits estrogen-like activity in bone and uterine tissue. These various MOAs are assumed to result from interactions between TAM and various proteins involved in the transcription of estrogen-responsive genes. The estradiol analogue fulvestrant, on the other hand, is a pure estrogen antagonist with no estrogenic properties. Having a greater affinity to the estrogen receptor than TAM, ICI competitively inhibits E2 binding to the ER and thereby inactivates transcription. Moreover, ICI-ER complexes are highly instable. Thus, ER down-regulation occurs due to ER protein degradation, resulting in a complete inhibition of estrogen signaling through ER. TAM and ICI can enter waste- and surface waters by being excreted by humans after ingestion, and sewage treatment works often fail in removing those substances. It reduced the percentages of ACs and increased the relative amount of the call type rasping, indicating a lowered sexual arousal of EE2 exposed males. This effect might be partly due to EE2 lowering testosterone levels, as it was shown for male X. laevis exposed to EE2 for 4 weeks, but it seems more likely that EE2 exhibits direct effects on vocalizations of male X. laevis. EE2 was also shown to alter spectral and temporal parameters of ACs of male X. laevis and these modifications of calling behavior were Masitinib VEGFR/PDGFR inhibitor suggested to be caused by alterations in the central vocal-motor pathway located in the central nervous system due to altered relations between endogenous androgens and estrogens or disruptions of genomic or non-genomic signalling pathways triggered solely by estrogens. Simultaneous exposure to environmentally relevant concentrations of EE2 and a 1000-fold TAM concentration resulted in fewer EE2 effects.