Expression of two Ae-related chains of the extracellular matrix glycoprotein laminin was induced as multipotent C3H10T1/2 mouse embryo fibroblasts differentiated into myoblasts and myofibers. C3H10T1/2 fibroblasts expressed the B1e (M(r) = 215,000) and B2e (M(r) = 205,000) laminin chains based on metabolic radiolabeling, immunoprecipitation, peptide mapping, and mRNA analysis. In contrast, myoblasts derived from C3H10T1/2 fibroblasts treated with DNA demethylating agents or transfected with the cDNA encoding MyoD expressed the Ae (M(r) = 400,000) and a novel Ae-related laminin chain (designated Ac3h, M(r) = 350,000) in addition to the B1e and B2e chains. Expression of the Ae and Ac3h chains paralleled the capacity for myofiber formation in six additional C3H10T1/2 myoblast clones with varied potentials for terminal differentiation and coincided with a switch in laminin isoforms from those of M(r) = 850,000 synthesized by C3H10T1/2 fibroblasts to those of M(r) = 900,000-950,000 synthesized by C3H10T1/2 myoblasts and myofibers. Cultures of mouse C2C12, mouse BC3H1, rat L6, and primary mouse myoblasts also synthesized the Ae, Ac3h, B1e, and B2e laminin chains. The results demonstrate that expression of the Ae and Ac3h laminin chains is associated with expression of MyoD and the mammalian myogenic differentiation program.