Some bacteria transferred during sexual activity cause substantial world-wide morbidity. In addition, the CS and distal urethras of healthy men at least episodically support bacterial communities. Lactobacillus spp. have been identified in urine and urethral swabs, and BV-associated taxa including Prevotella, Gardnerella and Sneathia are found in CS and urethral specimens from adult men. Although the role of bacteria in the male urethra is unknown, the CS microbiota has been hypothesized to mediate effects of circumcision on risk of HIV and other STI. A limitation in understanding the microbiota of the penis is the lack of data from healthy young men who have and or have not had partnered sexual experiences. These data would allow more thorough description of the microbiota of the urethra and CS, and could provide insight into changes associated with sexual exposures. To fill this gap, we collected urine and CS specimens from eighteen healthy 14–17 year old men with varied circumcision status and sexual histories. Sampling was repeated at monthly intervals to investigate stability of the microbiota over a three-month period. Bacteria were identified using multiple 16 S rRNA sequencing methods. Urine and corresponding CS specimens supported stable, but dissimilar microbiotas. Major urine taxa in most of the sexually experienced and inexperienced participants were members of the order Lactobacillialles. Finally, some bacteria were detected only in participants with histories of partnered sexual activity. The primer and barcode sequences were then trimmed from the remaining sequences using customized Perl scripts. Both the 16 S contigs from Sanger sequencing and 16 S raw sequences were classified with RDP Classifier v2.2 using a series of confidence cutoffs ranging from 0.6 to 0.9. For selected subset of genera member sequences were BLASTed against the SILVA database. Species-level PF-4217903 c-Met inhibitor assignments were decided based on near perfect alignments to top database matches and were manually examined for alignment quality. Multiple sequence alignments were produced using ClustalW with default parameters, and neighbor-joining phylogenic trees were constructed using PHYLIP. Principal coordinate analyses of microbial communities were performed using Unifrac. The above Unifrac analyses were also repeated by random sub-sampling from each specimen. Briefly, sequence reads were randomly extracted without replacement from each specimen and this procedure was repeated 100 times. The averaged sequence counts were used for Unifrac analyses. Microsoft EXCEL and customized scripts developed in the R statistical package were used for statistical analyses. To correct for multiple tests, false discovery rates were computed with the R package function qvalue. Wilcoxon’s rank sum test was used to assess comparisons of continuous variables between groups. The Wilcoxon’s signed rank test for paired data was used for comparisons between swab and urine samples from the same subject. Fisher’s exact test was used for comparisons of categorical variables between groups. Sørensen’s similarity index was calculated between either swab or urine specimens from different time points within each subject to assess intra-subject variability of microbiotas over time. Differences in the index between swab and urine samples were assessed using linear mixed-effects models.