The effect of fluconazole prophylaxis on the vaginal flora of 323 human immunodeficiency virus-infected women was evaluated in a multicenter, randomized, double-blind, placebo-controlled trial. Women with CD4 cell counts of < or = 300/mm3 received either 200 mg of fluconazole per week or placebo. Vaginal surveillance cultures were performed every 3 months. After a follow-up of 29 months, Candida albicans was recovered from 53% of patients receiving fluconazole and 68% of patients assigned placebo. Fluconazole was associated with a 50% reduction in the odds of being colonized with C. albicans but with higher rates for non-albicans Candida species. Candida glabrata was recovered from 40 women assigned fluconazole and 29 assigned placebo (relative odds, 1.96; 95% confidence interval, 0.98-3.94). Fluconazole had an early and persistent effect on the vaginal mycoflora, with the emergence of C. glabrata vaginal colonization within the first 6 months. The effect of fluconazole prophylaxis can be attributed to the reduction in vaginal C. albicans colonization; however, C. glabrata colonization rapidly supervened.