Monoclonal antibodies (McAb) to actin were prepared to analyze the assembly of actin isoforms in developing muscle cells in vitro. One of the antibodies (SkA-06) was specific for α-sarcomeric actin isoforms in skeletal and cardiac muscles, while the others recognized cytoskeletal (β, γ) actin isoforms in smooth muscle and non-muscle tissues as well as the sarcomeric (α) actins. Using SkA-06 and a polyclonal antibody (PcAb) specific for cytoskeletal actins, the subcellular localization of the actin isoforms was examined by immunocytochemical methods. While in developing young myotubes, cytoskeletal and sarcomeric actins were co-localized in nascent myofibrils or stress-fiber-like structures, sarcomeric actins predominated in striated myofibrils in more developed myotubes. When FITC-labeled cytoskeletal and sarcomeric actins were introduced into young myotubes by a microinjection method, the latter became detectable in striated structures sooner than the former but they were finally incorporated into striated myofibrils. These results suggest that α-actin(s) as well as β- and γ-actins can be incorporated into myofibrils, but α-actin(s) is assembled preferentially into myofibrils in developing muscle cells.