We have isolated and characterized two cDNA clones from whole rat stomach, pRV alpha A-19 and pRE gamma A-11, which are specific for the alpha-vascular and gamma-enteric smooth muscle isoactins, respectively. The rat gamma-enteric smooth muscle actin contains a single amino acid substitution of a proline for a glutamine at position 359 of the mature peptide when compared with the chicken gizzard gamma-actin sequence (J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979). Sequence comparisons of the 5' and 3' untranslated (UT) regions of the two smooth muscle actin cDNAs demonstrate that these regions contain no apparent sequence similarities. Additional comparisons of the 5' UT regions of the two smooth muscle actin cDNAs to all other known actin sequences reveal no apparent sequence similarities for the rat gamma-enteric isoactin within the 15 base pairs of sequence currently available, while the rat alpha-vascular isoactin contains two separate sequences which are similar to sequences within the 5' UT regions of the human and chicken alpha-vascular actin genes. A similar comparison of the 3' UT regions of the two smooth muscle actins demonstrates that the alpha-vascular isoactins do not contain the high degree of cross-species sequence conservation observed for the other isoactins and that the gamma-enteric isoactin contains an inverted sequence of 52 nucleotides which is similar to a sequence found within the 3' UT regions of the human, chicken, and rat beta-cytoplasmic isoactins. These observations complicate the apparent cross-species conservation of isotype specificity of these domains previously observed for the other actin isoforms. Northern blot analysis of day 15 rat embryos and newborn, day 19 postbirth, and adult rats demonstrates that the day 15 rat embryo displays low to undetectable levels of smooth muscle isoactin mRNA expression. By birth, the stomach and small intestine show dramatic increases in alpha-vascular and gamma-enteric actin expression. These initially high levels of expression decrease through day 19 to adulthood. In the adult rat, the uterus and aorta differ in their content of smooth muscle isoactin mRNA. These results demonstrate that the gamma-enteric and alpha-vascular isoactin mRNAs are coexpressed to various degrees in tissues which contain smooth muscle.