We undertook this study to identify the risk factors and prevalence of Cryptosporidium spp. in HIV-positive and HIV-negative diarrhea patients in the Alice rural settlement in the Eastern Cape Province of South Africa. A total of 180 stool specimens (35 HIV-positive diarrhea, 125 HIV-negative diarrhea patients, and 20 apparently healthy subjects) were screened for cryptosporidiosis using an ELISA-based approach. Sociodemographic information, water supply, and animal contact were recorded for diarrhea-positive patients. The data were analyzed using Pearson's chi2-test and Fisher's exact test. Cryptosporidium antigen was detected in 122 of 180 specimens (overall prevalence=67.8%). In HIV-positive diarrhea patients, the age groups 31-43 years (mean age 36.5 years) and 70-82 years (mean age 75.8 years) had a higher prevalence (100%) of the antigen than age groups 18-30 years (mean age 23.2 years) and 83-95 years (mean age 88.8 years) (50.0%). In HIV-negative diarrhea patients, the prevalence was highest (87.5%) at ages 18-30 years (mean age 23.2 years) and lowest (35.7%) at ages 83-95 years (mean age 88.8 years). Cryptosporidium antigenemia was slightly higher in females (78.2%, mean age 46.7 years) than in males (71.1%, mean age 42.6 years), but the difference was not significant (p>0.05). No apparently healthy control subject was infected with Cryptosporidium. HIV-negative patients had a significantly higher prevalence of antigen than HIV-positive patients, with farm animals considered a possible risk factor. In HIV-positive diarrhea patients, the prevalence peak was detected in more low income patients (85.7%) than in high income patients (32%). The high infection rate of specific groups was associated with exposure to a contaminated water supply. The results indicate that Cryptosporidium infection is highly prevalent in adult fecal specimens from the Nkonkobe Municipality, an indication of active infection that is likely to emerge as a major human pathogen in this locality owing to socioeconomic changes that favor transmission.