Although Pneumocystis carinii is the most common opportunistic pathogen infecting individuals with AIDS, very little is known of the basic biology of the organism. We have examined the ribosomal RNA (rRNA) and the DNA encoding it (rDNA) in P. carinii in an attempt to clarify its taxonomic position and to begin to study its genetic processes. Electrophoretic analysis showed that the sizes of the P. carinii rRNAs are quite similar to the sizes of the corresponding rRNAs from Saccharomyces cerevisiae. Direct sequence analysis of approx. 60% of the 18S small subunit-rRNA (Ss-rRNA) confirmed that its sequence is similar to that of yeast-like fungi and that a putative group-I intron previously observed in the 18S rDNA is, in fact, excised from the mature rRNA. PCR analysis of the intron in P. carinii genomic DNA showed that each of the multiple rDNA genes bears the group-I intron and in vitro transcripts of the intron autocatalytically excise from the rRNA primary transcript in the presence of GTP. Finally, analogues of GTP inhibit the self-splicing reaction, indicating that the guanosine-binding site of the intron closely resembles that of other well-characterized group-I introns. Since no group-I introns have been found in higher eukaryotes, this self-splicing process represents a viable target for chemotherapy of P. carinii pneumonia (PCP).