We have utilized cloned actin genes from Drosophila melanogaster and from chicken to isolate 12 actin gene fragments from a human DNA library. Each of these 12 clones was shown to contain actin coding regions by its ability to selectively hybridize to human actin mRNA as assayed by in vitro translation. The translation product was judged to be actin on the basis of its comigration with authentic actins when electrophoresed on one- and two-dimensional NaDodSO4/polyacrylamide gels and on the basis of its partial proteolysis products. Determination of the sizes and order of the fragments generated by restriction endonuclease digestion of each of these recombinant phages allows us to conclude that they are nonallelic and are from nonoverlapping regions of the genome. We have used these cloned human actin genes and the Drosophila and chicken actin gene clones to show that the human genome contains 25-30 EcoRI fragments homologous to actin genes and that, among three nonconsanguineous individuals tested, none of these fragments exhibit length or restriction-site polymorphism.