BACKGROUND: In 1997, a survey in Kisumu found a prevalence of HIV infection among female sex workers (FSW) of 75%. Only 50% reported using a condom with the last client. In 2008, we conducted another survey to collect data to inform an intervention targeting FSW in Kisumu. METHODS: In 2008 FSW were recruited by respondent-driven sampling. Women completed a questionnaire and were tested for HIV and other sexually transmitted infections (STIs). Multiple logistic regression analysis was done to explore factors associated with HIV-infection, and with condom use. Prevalence of HIV infection was compared in the two surveys from 1997 and 2008. Multivariate analysis was used to assess whether a change in HIV prevalence between the two surveys could be explained by changes in socio-demographic characteristics and/or behavioral factors. RESULTS: 481 FSW participated in the 2008 study. HIV prevalence was 56.5% (95% CI 52.0-61.6). Factors independently associated with HIV were age older than 29 years; being a widow; STI treatment in the past year; herpes simplex virus Type-2 infection; bacterial vaginosis; and trichomoniasis. Condom use with last client was reported by 75.0% (95% CI 70.9-78.9). Predictors of condom use with the last client were age older than 29 years; higher price paid by last client; ever having been tested for HIV. Predictors of unprotected sex were being drunk during last sex act; usually having sex during menses; and STI treatment in the past year. The odds ratio of HIV infection associated with year of survey was 0.49 (95% CI 0.33-0.75) after adjusting for socio-demographic and behavioral factors. CONCLUSIONS: The prevalence of HIV among FSW in Kisumu was found to be lower in 2008 than in 1997, while reported condom use was higher. However, access to HIV/STI prevention and care services needs to improve to further decrease HIV transmission between FSW and their clients.