Sequence analysis of a polymerase chain reaction (PCR)-amplified 298-bp region of the Cryptosporidium parvum 18S rRNA gene was carried out on 10 human and 9 animal isolates. Eight of the 9 animal isolates and 3 human isolates displayed the recognition sequence TATATTT, whereas 7/10 human isolates exhibited the recognition sequence TTTTTTTTTTT. Sequence analysis of the ninth animal isolate, which was recovered from a Koala, revealed this isolate to be different from both human and animal isolates. The AT richness of the rDNA recognition sequences rendered them unsuitable for primer design and therefore a diagnostic randomly amplified polymorphic DNA fragment previously developed in our laboratory was also sequenced. Analysis of 2 human and 2 animal isolates again revealed distinct differences between animal and human isolates. On the basis of this sequence information, diagnostic primers were designed that could directly differentiate between animal and human isolates on the basis of the size of the PCR product. The ability to differentiate directly between human and animal isolates has important implications for studies of the transmission and zoonotic potential of this organism. These results also raise further doubts about the uniformity of the species C. parvum.