BACKGROUND: Cotrimoxazole is recommended for prevention of opportunistic infections in symptomatic HIV patients in sub-Saharan Africa. METHODS: We examined the feasibility and effectiveness of daily cotrimoxazole prophylaxis in a well-established cohort of HIV-infected adults attending clinics in Entebbe, Uganda. We compared mortality and morbidity rates for 12 months before and after the introduction of cotrimoxazole. RESULTS: Between August 2000 and February 2002, 94% of cohort members were enrolled onto cotrimoxazole prophylaxis. Revisits were scheduled every 4 weeks to replenish pills; patients attended 61% of revisits. The main reasons for nonenrollment and defaulting were lack of transport, being away from home, and sickness. Drug-related adverse events, mainly itching and rash, were seen in 4% of participants. Although bacterial resistance rate to cotrimoxazole was high, the adjusted mortality incidence rate ratio was significantly reduced after the introduction of cotrimoxazole (0.76; 95% confidence interval, 0.60-0.96; P = 0.020). Overall febrile events and morbidity rates were unchanged after the introduction of cotrimoxazole, but the incidence of malaria was reduced (incidence rate ratio, 0.31; 95% confidence interval, 0.13-0.72). CONCLUSIONS: Cotrimoxazole prophylaxis can be introduced into routine HIV clinic activities and is associated with a reduction in overall mortality and malaria morbidity, even in an area with high bacterial resistance. These results reinforce the need for large-scale provision of cotrimoxazole prophylaxis for all HIV-positive patients in developing countries.