The study objective was to investigate whether sex influences 3′-deoxy-3′- 18 F-fluorothymidine ( 18 F-FLT) uptake and tissue distribution in mouse models of cancer. Methods: 18 F-FLT biodistribution was measured in 3 strains of male and female mice (129S6/SvEv, athymic nude, and BALB/c). 18 F-FDG biodistribution was measured for comparison. 18 F-FLT uptake was also measured in female 129S6/SvEv mice bearing estrogen-dependent SSM3 mouse mammary tumors, male athymic nude mice bearing androgen-dependent CWR22 prostate cancer xenografts, and male and female athymic nude mice bearing estrogen-independent MDA-MB-231 human breast cancer xenografts. Ki-67 expression was assayed by immunohistochemistry. PET/CT imaging was performed to visualize 18 F-FLT biodistribution and to determine pharmacokinetics. Results: Greater 18 F-FLT activity was observed in blood, liver, muscle, heart, kidney, and bone in female than male mice. Pharmaco-kinetic analysis demonstrated higher early renal 18 F-FLT activity and greater accumulation of 18 F-FLT in the urinary bladder in male than female mice. The differential pattern of 18 F-FLT biodistribution between the sexes seen with 18 F-FLT was not observed with 18 F-FDG. Increased tumoral 18 F-FLT uptake compared with muscle was observed in both the SSM3 mammary tumors (2.4 ± 0.17 vs. 1.6 ± 0.14 percentage injected dose [%ID]/g at 2 h after injection, P = 0.006) and the CWR22 prostate cancer xenografts (0.34 ± 0.08 vs. 0.098 ± 0.033 %ID/g at 2 h after injection, P = 0.03). However, because of higher nonspecific muscle uptake in female mice, tumor-to-muscle uptake ratios were greater for CWR22 tumors than for SSM3 tumors (4.2 ± 0.78 vs. 1.5 ± 0.049 at 2 h after injection, P = 0.008). Sex-dependent differences in 18 F-FLT uptake were also observed for MDA-MB-231 xenografts (tumor-to-muscle ratio, 7.2 ± 0.9 for female vs. 16.9 ± 8.6 for male, P = 0.039). Conversely, greater tumoral Ki-67 staining was observed in female mice (71% ± 3% for female vs. 54% ± 2% for male, P = 0.009), and this finding more closely matched the relative differences in absolute 18 F-FLT tumor uptake values (4.5 ± 0.99 %ID/g for female vs. 1.9 ± 0.30 %ID/g for male, P = 0.03). Conclusion: Depending on whether female or male mice are used, differences in biodistribution and nonspecific tissue uptake can adversely affect quantitative measures of 18 F-FLT uptake. Thus, sex is a potential variable to consider in defining quantitative imaging metrics using 18 F-FLT to assess tumor proliferation.