A cross-sectional serosurvey for markers of prior Cryptosporidium infection was conducted among homosexual or bisexual males infected with human immunodeficiency virus (HIV); of 262 individuals approached, 236 (90%) agreed to participate. Serological response to two Cryptosporidium antigens was measured using a Western blot assay. The intensity or detection of serological responses to two Cryptosporidium antigens was not associated with CD4 cell counts or tap water consumption. A number of sexual practices were related to increased serological response for only the 27-kDa marker, including having had sex within the past 2 years, having anal sex and having had a larger number of sex partners during the past 2 years. Attending a spa or sauna was related to serological response to both the 27-kDa and 17-kDa markers. Based on these results, activities related to sexual activity appear to be a significant risk factors for prior Cryptosporidium infection.