Androgens and the androgen receptor (AR) play important roles in virilization, spermatogenesis, and sexual behavior in vertebrates. An understanding of the distribution and levels of expression of the ARs on the cellular and tissue level demonstrates the pattern of responsiveness to the androgenic hormones in a given organism. In this study, a fragment of the AR gene was cloned and sequenced from the bonnethead shark, Sphyrna tiburo, an elasmobranch species with a well-defined annual reproductive cycle. Acquiring this gene sequence facilitated the construction of species-specific AR polymerase chain reaction (PCR) primers and species-specific AR mRNA probes that were used to screen reproductive tissues for evidence of AR gene expression using reverse transcription (RT)-PCR and in situ hybridization (ISH), respectively. The RT-PCR screens demonstrated AR gene expression in the testes, epididymides, seminal vesicles, and claspers of male sharks. The use of relative PCR revealed that these organs have variable levels of AR gene expression that significantly differ with the stage of the shark's seasonal reproductive cycle. ISH results localized the AR RNA in the interstitial cells, Sertoli cells, and developing sperm of the testes, and mature spermatozoa within the seminal vesicles and the epididymides. Immunocytochemical methods used to detect the AR protein using a rabbit polyclonal antibody, PG-21, produced comparable results in the shark testes but did not yield positive results in the seminal vesicles or the epididymides. However, the Leydig gland, whose secretions contribute to the seminal fluid, demonstrated consistent AR immunoreactivity. Results of ICC in male and female embryos of S. tiburo revealed AR protein in the developing kidney but not in the embryonic reproductive structures. By characterizing AR distribution in the reproductive tract of male S. tiburo, this study provides the basis for future research on the direct and indirect effects of androgenic hormones in this species.