The gene coding for actin from Phaffia rhodozyma was cloned and sequenced. The Phaffia actin gene contains four intervening sequences and the predicted protein consists of 375 amino acids. The structural features of the Phaffia actin introns were studied and compared with actin introns from seven fungi and yeasts with ascomycetous and basidiomycetous affinity. It was shown that the architecture of the Phaffia introns most resembles that of the basidiomycete Filobasidiella neoformans (perfect stage of Cryptococcus neoformans), whereas least resemblance occurs with the ascomycetous yeasts. Based on the intron structure, the ascomycetous yeasts can be accommodated in one group in that their splice site sequences are very similar and show less homology with the other fungi investigated, including Phaffia. It was demonstrated that the Phaffia actin introns cannot be spliced in Saccharomyces cerevisiae, which shows that the differences found in intron structure are significant. Alignment of the Phaffia actin gene with the actin sequences from the yeasts and fungi investigated showed a high level of homology both on the DNA level and on the protein level. Based on these alignments Phaffia showed highest homology with F. neoformans and both organisms were accommodated in the same cluster. In addition, the actin gene comparisons also supported the distant relationship of Phaffia with the ascomycetous yeasts. These results supported the usefulness of actin sequences for phylogenetic studies.