Diverse proteins that are 35% to 55% identical to actins have been discovered recently in yeasts, nematodes, and vertebrates. In order to study these proteins systematically and relate their functions to those of conventional actins, we are isolating the corresponding genes from the genetically tractable eukaryote, Drosophila melanogaster. Here we report the isolation and partial characterization of a Drosophila homologue of the Schizosaccharomyces pombe act2 gene. Degenerate oligonucleotide primers specifying peptides that are highly conserved within the actin protein superfamily were used in conjunction with polymerase chain reaction (PCR) to amplify a portion of the Drosophila gene that we have named actr66B. The corresponding full-length cDNA sequence encodes a protein of 418 residues that is 65% identical to the product of the S. pombe act2 gene, 80% identical to the bovine act2 homologue, but only 48% identical to the principal Drosophila cytoplasmic actin encoded by the Act5C actin gene. Alignment of the yeast, bovine, and Drosophila actin-related proteins shows that they have four peptide insertions, relative to conventional actins, three of which are well placed to modify actin polymerization and one that is likely to perturb the binding of myosin. Locations of two of the five actr66B introns are conserved between Drosophila and yeast genes, further attesting that they evolved from a common ancestor and are likely to encode proteins having similar functions. We demonstrate that the Drosophila gene is located on the left arm of chromosome 3, within subdivision 66B. Finally, we show by RNA blot-hybridization that the gene is expressed at low levels, relative to conventional nonmuscle actin, in all developmental stages. From these and other observations we infer that the actr66B protein is a minor component of all cells, perhaps serving to modify the polymerization, structure, and dynamic behavior of actin filaments.