Ascidian embryos develop rapidly into tadpole larvae containing striated tail muscle cells. We have isolated and characterized five actin cDNA clones from a Styela clava tailbud-stage library. The nucleotide sequences of these clones and genomic Southern blot analysis indicate that they represent at least four different muscle actin genes, which are designated ScTb1, ScTb24, ScTb30, and ScTb12/34. The derived protein sequences of these genes indicate that they encode the same alpha-muscle actin which has features related to each of the three different classes of vertebrate alpha-muscle actins. Northern and in situ hybridization with probes prepared from the 3' untranslated region (UTR) of several of the ScTb clones showed that these muscle actin genes are expressed in different temporal and spatial patterns during development. ScTb1 was detected in eggs, embryos, and adults, ScTb24 and ScTb12/34 were detected in embryos and adults, and ScTb30 was detected only in embryos. The maternal transcripts disappeared shortly after fertilization and zygotic mRNAs were first detected during gastrulation and continued to accumulate during subsequent tail muscle differentiation. ScTb30 mRNA, which is expressed in the embryo, peaks during the tailbud stage and is present at low levels in the tadpole larva. In contrast, ScTb1, ScTb24, and ScTb12/34 mRNAs, which are expressed in embryos and adults, peak during the late tailbud stage and are present in substantial quantities in the larva. The ScTb24 gene was detected only in tail muscle cells, whereas the ScTb30 gene was detected in embryonic tail muscle, mesenchyme, epidermal, and neural cells. The ScTb24 mRNA also accumulates primarily in vascular tissue in the branchial sac and mantle of adults. The existence of a gene family encoding the same alpha-muscle actin isoform is unique among the chordates and may function to maximize muscle actin production during the rapid differentiation phase of ascidian larval muscle cells.