ADP-ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are involved in intracellular vesicular transport. Based on size, phylogenetic analysis, amino acid identity, and gene structure, mammalian ARFs fall into three classes (class I, ARF1, -2, and -3; class II, ARF4 and -5; class III, ARF6). A class I ARF had been identified in Drosophila melanogaster. To search for ARFs of other classes in Drosophila, polymerase chain reaction-based techniques were used, resulting in cloning of Drosophila ARF (dARF) II and dARF III with deduced amino acid sequences similar to those of class II and class III mammalian ARFs, respectively. The three Drosophila ARF genes map to different chromosomes and the coding regions have different splicing sites. dARF II mRNA, like ARF I mRNA, is fairly uniformly distributed throughout adult flies, whereas dARF III mRNA is significantly more abundant in heads than in legs or bodies. Recombinant dARF II and dARF III have biochemical and immunological properties similar to those of human ARF5 (hARF5) and hARF6, respectively. These observations are consistent with the conclusion that the three classes of ARFs are present in non-mammalian as well as mammalian species.