Changes of actin isomers during development and differentiation of chicken gizzard were investigated by polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis with SDS-polyacrylamide gels in the presence of urea as the second dimension clearly separated three actin isomers which appear during development of the smooth muscle. The three actin isomers change the relative concentrations during development as follows: gizzard-type gamma-actin begins to be synthesized late on the 7th day of embryogenesis and increases in amount until hatching, nonmuscle-type gamma-actin exists only at earlier stages (before 15 days of embryogenesis), and the amount of beta-actin increases in proportion to the decrease of nonmuscle type gamma-actin, the amount of nonmuscle actin in gizzards then becoming constant. Actin composition of gizzard before 7 days of embryonic age was nonmuscle type and consisted of beta-actin and nonmuscle-type gamma-actin. These observations indicate that developmental process of gizzard smooth muscle cells are classified as three stages: nonmuscle, intermediate and smooth muscle stages.