The actin gene from yeast Saccharomyces carlsbergensis was cloned in Escherichia coli and its complete nucleotide structure was determined. A comparison of its DNA sequence with that of the related yeast species Saccharomyces cerevisiae revealed that the coding as well as the 5'- and 3'-untranslated regions are identical. The intron, although at the same location in the two genes, differs in three positions. There is one deletion (or insertion), one transition, and one transversion. These are clustered within and around an oligo(dA) stretch of 14 (or 13) residues. Our observations identify the intron as the fastest evolving segment of this eukaryotic gene.