Prostate cancer (PC) is both an age- and an androgen-dependent disease. Paradoxically, systemic levels of androgens decline with age as the risk of PC rises. While there is no correlation between systemic androgen levels and the risk of PC, systemic androgen levels do not reflect the levels of androgens in prostate tissue. In metastatic PC, changes in the androgen biosynthesis pathway during hormone therapy result in increased levels of androgens in cancer tissue and contribute to continued androgen receptor (AR) signaling. It is possible that similar changes occur in normal prostate tissue as androgen levels decline with age and that this contributes to tumorigenesis. In the present study, we sought to determine whether the rat prostate is able to maintain functional levels of androgens despite low serum testosterone levels. Rats were castrated and implanted with capsules to achieve castrate, normal, sub-physiological, and supra-physiological levels of testosterone. After 6 weeks of treatment, LC–MS/MS was used to quantify the levels of testosterone and dihydrotestosterone (DHT) in the serum and prostate tissue. Quantitative RT-PCR was used to quantify the expression of genes involved in the androgen/AR signaling axis. Despite significantly different levels of testosterone and DHT being present in the serum, testosterone and DHT concentrations in prostate tissue from different testosterone-treatment groups were very similar. Furthermore, the expression of androgen-regulated genes in the prostate was similar among all the testosterone-treatment groups, demonstrating that the rat prostate can maintain a functional level of androgens despite low serum testosterone levels. Low-testosterone treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining functional androgen levels.