Several species of Cryptosporidium have been associated with infection. Cryptosporidium parvum and Cryptosporidium hominis are the main agents of cryptosporidiosis in humans. Stool samples from 108 Cryptosporidium-infected patients were submitted to PCR-RFLP analysis for a 553-bp fragment of Cryptosporidium oocyst wall protein (COWP) gene and an 826-864 bp fragment of the small-subunit ribosomal RNA (SSU-rRNA) gene. Ninety-two patients were immunocompetent children and 16 were HIV-infected adults. C. hominis was detected in 69 patients (59 immunocompetent and 10 HIV-infected); C. parvum, in 34 patients (28 immunocompetent and 6 HIV-infected); and C. meleagridis and C. felis in one patient each (both immunocompetent children). Three samples yielded negative results. C. parvum was significantly more frequent in children from rural areas than in those of urban residence (p=0.010). As far as we know, this is the first surveillance study about the molecular characterization of Cryptosporidium in humans performed in Spain. The finding of zoonotic species infecting humans calls for further research on this subject.