We hypothesized that imaging-based assessment of cellular proliferation in prostate cancer may improve tumor characterization. We therefore evaluated the biodistribution and effect of androgen on tumor uptake of the cellular proliferation imaging marker [(18)F]-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ((18)F-FMAU) in xenograft mouse models of human prostate cancer. Castrated and noncastrated athymic male mice were implanted with androgen-independent PC3 and androgen-sensitive CWR22 human prostate cancer cells. Dynamic micro-positron emission tomography (PET)/computed tomography was performed for 1 hour followed by 10-minute static scans at 2 and 3 hours. Animals were sacrificed after imaging for biodistribution studies and immunohistochemical staining of tumors for androgen receptor and Ki-67/MIB expression. (18)F-FMAU uptake was significantly higher in all major organs of the castrated animals in comparison with noncastrated mice, with the highest uptake in liver and the lowest uptake in muscle and bone. When compared to PC3 tumors, CWR22 xenografts showed significantly higher tumor to muscle (2.56 ± 0.30 vs 1.99 ± 0.30, p  =  .008) and tumor to liver (1.72 ± 0.12 vs 1.26 ± 0.17, p  =  .0003) uptake ratios in the noncastrated animal at the 3-hour time point. Androgen receptor and Ki-67/MIB expressions were higher in CWR22 than in PC3 xenografts. Our initial preclinical observations suggest that there may be an association between androgen signaling and thymidine metabolism and that (18)F-FMAU PET may be useful in prostate tumor characterization.