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Feces from 34 species of ruminants housed at the Lisbon Zoo was examined for Cryptosporidium spp. oocysts. Three hundred and eighty-eight samples were analyzed. Three hundred and eighty species-specific group fecal samples were collected monthly, from September 1998 until August 1999, along with eight individual specimens from eight neonates. All samples were examined by four different techniques: microscopic observation of direct and concentrated fecal smears, staining with modified Ziehl–Nielsen, immunofluorescent assay, and immunoenzymatic assay. The prevalence of infection was 3.6%. Five neonates with diarrhea were infected. Cryptosporidial oocysts were shed more frequently during winter months. Some facilities may have permitted oocysts to remain viable, possibly contributing to cryptosporidial transmission between animals.