The sequential appearance of mRNAs for smooth, cardiac, and skeletal alpha-actin has been described during development of the chicken heart (Ruzicka, D.L., and R.J. Schwartz 1988 J. Cell Biol., 107:2575-2586). To assess whether this reflects the deposition of corresponding isoproteins, we have immunocytochemically localized smooth and sarcomeric (cardiac and skeletal) alpha-actin in Hamburger-Hamilton (H-H) stage 7-18 embryos using monoclonal antibodies. Within the developing embryo at stage 9-, smooth muscle alpha-actin was exclusively detected in the developing heart, upon fusion of the endocardial tubes; sarcomeric alpha-actin was observed later (stage 9). By the onset of contraction at stage 10+, intense immunostaining of both smooth and sarcomeric isoproteins was observed in the ventricle; at this time smooth muscle alpha-actin was also detected in splanchnic mesoderm of the pre-vitelline area, in a cellular layer adjacent to the only embryonic cells that exhibited factor VIII (von Willebrand factor) antigens. Double immunostaining of the myocardium at stage 11, at which time striations were first detected, revealed the co-existence of smooth and sarcomeric actin in developing sarcomeres. Intense expression of sarcomeric actin continued in the heart after stage 11, whereas smooth muscle alpha-actin was down-regulated in the ventricle and became regionalized to the inflow and outflow tracts. As expected, smooth muscle alpha-actin was detected around intra- and extra-embryonic vascular structures at later developmental stages, while sarcomeric actin was observed in somites.