The actin gene family of the sea urchin Strongylocentrotus purpuratus was analyzed by the genome blot method, using subcloned probes specific to the 3' terminal non-translated actin gene sequence, intervening sequence and coding region probes. We define an actin gene subtype as that gene or set of genes displaying homology with a given 3' terminal sequence probe, when hybridized at 55 degrees C, 0.75 M-Na+. By determining the often polymorphic restriction fragment band pattern displayed in genome blots by each probe, all, or almost all of the actin genes in this species could be classified. Our evidence shows that the S. purpuratus genome probably contains seven to eight actin genes, and these can be assigned to four subtypes. Studies of the expression of the genes (Shott et al., 1983) show that the actin genes of three of these subtypes code for cytoskeletal actins (Cy), while the fourth gives rise to a muscle-specific actin (M). We denote the array of S. purpuratus actin genes indicated by our data as follows. There is a single CyI actin gene, two or possibly three CyII genes (CyIIa, CyIIb, and possibly CyIIc), three CyIII actin genes (CyIIIa, CyIIIb, CyIIIc), and a single M actin gene. Comparative studies were carried out on the actin gene families of five other sea urchin species. At least the CyIIa and CyIIb genes are also linked in the Strongylocentrotus franciscanus genome, and this species also has a CyI gene, an M actin gene and at least two CyIII actin genes. It is not clear whether it also possesses a CyIIc actin gene, or a CyIIIc actin gene. The genome of a more closely related congener, Strongylocentrotus dröbachiensis, includes 3' terminal sequences suggesting the presence of a CyIIc gene. In S. franciscanus and S. dröbachiensis the first intron of the CyI gene has remained homologous with intron sequences of both the CyIIa and CyIIb genes, indicating a common origin of these three linked cytoskeletal actin genes. Of the four S. purpuratus 3' terminal subtype probe sequences only the CyI 3' terminal sequence has been conserved sufficiently during evolution to permit detection outside of the genus Strongylocentrotus. An unexpected observation was that a sequence found only in the 3' untranslated region of the CyII actin gene in the DNA of S. dröbachiensis and S. purpuratus is represented as a large family of interspersed repeat sequences in the genome of S. franciscanus.