The process of urbanization occurring in many developing countries may have consequences for reproductive endocrine function. Here, we test predictions concerning variation in South African male testosterone levels among subgroups across an urbanization gradient representing differences in both geography and socioeconomic status. Subjects included 364 males aged between 20 and 82 comprising a cross-sectional study conducted between 1996 and 1998. Testosterone levels were measured from serum samples obtained between 08:00 and 11:00. In ANCOVA analysis, male testosterone levels differed significantly along this rural-to-urban gradient, with members of the most urban group having higher testosterone levels than groups of farmers and inhabitants of informal housing areas adjacent to towns. Testosterone levels declined with age and were negatively related to body mass index (BMI). Testosterone levels did not differ according to HIV status. Further exploratory ANCOVA analyses revealed that physical activity levels, depression, affect, and hostility were not significantly associated with variation in testosterone levels. These data help document causes of variation in male testosterone levels in a context of urbanization and may have implications for clinical outcomes such as the development of a male hormonal contraceptive or prostate cancer. Am. J. Hum. Biol. 18:123–132, 2006. © 2005 Wiley-Liss, Inc.