We have constructed isotype-specific subclones from the 3' untranslated regions of alpha-skeletal, alpha-cardiac, beta-cytoskeletal, and gamma-cytoskeletal actin cDNAs. These clones have been used as hybridization probes to assay the number and organization of these actin isotypes in the human genome. Hybridization of these probes to human genomic actin clones (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981; Engel et al., Mol. Cell. Biol. 2:674-684, 1982) has allowed the unambiguous assignment of the genomic clones to isotypically defined actin subfamilies. In addition, only one isotype-specific probe hybridizes to each actin-containing gene, with a single exception. This result suggests that the multiple actin genes in the human genome are not closely linked. Genomic DNA blots probed with these subclones under stringent conditions demonstrate that the alpha-skeletal and alpha-cardiac muscle actin genes are single copy, whereas the cytoskeletal actins, beta and gamma, are present in multiple copies in the human genome. Most of the actin genes of other mammals are cytoplasmic as well. These observations have important implications for the evolution of multigene families.