Using heterologous probing of a genomic library, we have cloned and sequenced the actin gene from the pathogenic yeast Cryptococcus neoformans. The actin gene is 1371 bp in length, and exists as a single copy, as is the case for all fungi studied to date. The locations of the introns in the C. neoformans actin gene are unique among all other known actin genes, and the deduced coding sequence results in a 375 amino acid chain with very high homology to other actins. A phylogenetic tree comprising 31 actin-coding sequences from a wide variety of organisms shows that the C. neoformans actin gene is grouped on a distinct branch together with all other known fungal actin sequences. The availability of the C. neoformans actin gene will aid future phylogenetic and molecular studies of this important human pathogen.