Our objective was to determine the prevalence and risk factors for HIV infection among female sex workers in Johannesburg, South Africa. A cross-sectional survey of female sex workers was conducted using interviewer-administered questionnaires. Prevalent sexually transmitted infections including HIV were evaluated through standard laboratory testing. HIV infection was identified in 137 (46.4%) of 295 subjects tested. Increasing frequency of condom use was significantly negatively associated with HIV infection (odds ratio [OR] for moderate use = 0.21; 95% confidence interval [CI]: [0.09, 0.50]; OR for high use = 0.14; 95% CI: [0.06, 0.34]). Sex workers aged > or = 29 years reported significantly different patterns of behaviour than younger workers. Among women aged > or = 29, a negative association with HIV infection (OR = 0.16; 95% CI: [0.07, 0.38]) was found, but only among those not infected with Neisseria gonorrhoeae. Older women in the Johannesburg sex industry may have adaptive behavioural strategies besides condom usage which reduce their risk of acquiring HIV. However, older sex workers with gonorrhoea constitute a high-risk subgroup.