The nucleotide sequences of the 16S-like, 5.8S and 23S-like rDNAs from the two varieties of Cryptococcus neoformans, C. neoformans var. neoformans and C. neoformans var. gattii, were determined. The rRNA locus has the typical eukaryote organization of 16S-5.8S-23S with the 16S-like and 5.8S rRNA genes separated by a 124-nucleotide spacer and the 5.8S and 23S-like rRNA genes separated by a 187-nucleotide spacer in each strain. The C. neoformans var. neoformans and C. neoformans var. gattii 16S-like, 5.8S and 23S-like rRNAs are, respectively 1802, 158, and 3358 nucleotides in length and share > 99% nucleotide sequence identity, a finding which strongly supports the present taxonomic classification of two varieties within a species. Comparative structure analysis was used to construct secondary-structure models for the deduced 16S-like and 5.8S-23S-like rRNA sequences, which are similar to those of other fungal rRNAs. The C. neoformans 16S-like and 23S-like rRNA sequences were aligned with other eukaryote sequences based on secondary and higher-order structures predicted by comparative structure analysis for phylogenetic analysis. There was good correspondence between the 16S-like and 23S-like derived phylogenetic trees. The closest known fungal relative is Trichosporon beigelii. Southern blot analysis revealed one C. neoformans strain with two types of DNA repeats coding for rRNA which differed in size by about 1000 bp. Restriction fragment length polymorphisms in the rDNA locus provide useful markers for the study of epidemiology and pathogenesis of C. neoformans infections.