The epidemiology of human microsporidiosis is poorly understood and environmental factors affecting transmission of the organism have not been fully elucidated. Temporal variation in the prevalence of microsporidia in the stool of patients with human immunodeficiency virus (HIV) infection and diarrhea was studied to evaluate the role of water-borne transmission. From January 1993 to December 1996, 8,439 stools from HIV-infected individuals were examined for microsporidia spores in southern California. Yearly positivity rates were 8.8% in 1993, 9.7% in 1994, 6.6% in 1995, and 2.9% in 1996. An analysis for linear trend showed a statistically significant decrease in stool positivity rates over time (chi2 = 81.9, P = 0.001). No significant seasonal variation in the prevalence of microsporidiosis was seen over that time period. These results suggest the constant presence of microsporidia in the environment, rather than a seasonal association with recreational water use or seasonal contamination of the water supply, and a real decrease in yearly prevalence of microsporidia related diarrhea. Factors related to a progressive decrease in prevalence are subjects of future investigation.