Little is currently known regarding the ontogeny of smooth muscle tissues during normal mammalian development. The α-smooth muscle and γ-smooth muscle isoactins have been shown to be excellent molecular markers of smooth muscle cell phenotype. This study characterizes both the temporal and spatial patterns of α-smooth muscle and γ-smooth muscle isoactin expression in the developing mouse. In situ analysis was performed on serial sections of whole mouse embryos on embryonic day 9, 11, 13, 15, and 17 using α-smooth muscle and γ-smooth muscle isoactin-specific riboprobes. Distinct temporal and spatial patterns of α-smooth muscle and γ-smooth muscle isoactin gene expression were observed in the developing gastrointestinal tract, urogenital tract, respiratory tract, and vascular system. Independent expression of the α-smooth muscle isoactin was observed during the early stages of skeletal, cardiac, and smooth muscle myogenesis as well as in a novel subset of distinct organs including the postanal component of the hindgut, allantois, and primitive placenta. The results of this study indicate that distinct cellular phenotypes are involved in smooth muscle myogenesis and suggest that organ-specific mechanisms might exist for the initiation of smooth muscle development in vivo. In addition, the pattern of independent α-smooth muscle isoactin expression observed in this study provides novel information regarding the early stages of hindgut and placental development, and suggests that a common functional phenotype may be associated with the early stages of skeletal, cardiac, and smooth muscle myogenesis. © 1995 wiley-Liss, Inc.