Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools.Several Cryptosporidium species can cause severe acute diarrhea in humans and animals (5, 6, 13). Human cryptosporidiosis is usually self-resolving within a few days, but immunocompromised patients can develop life-threatening complications. Outbreaks due to drinking water contamination continue to occur (7,10,18,32), and there is no effective treatment, making cryptosporidiosis a major public health issue and economic problem. Diagnosis is generally based on microscopic detection of oocysts in stools, but this offers no information on the infecting species and is not suited to epidemiological investigations.The following 13 Cryptosporidium species are currently accepted, on the basis of host specificity, pathogenesis, morphology (9) and genotyping (8,16,21): Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium wrairi, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium andersoni, and Cryptosporidium muris as infecting mammals; Cryptosporidium baileyi, Cryptosporidium meleagridis, and Cryptosporidium galli as infecting birds; Cryptosporidium serpentis and Cryptosporidium saurophilum as infecting reptiles; and Cryptosporidium molnari as infecting fish (36). Recent phylogenetic analyses based on sequencing of the small subunit rRNA gene (18S rRNA) (21,22,24,33,34), the hsp 70 gene (31), or other housekeeping or structural genes (24,(28)(29)(30) show a complex multispecies organization of the g...