Adultation is a heterochronic mode of development in which adult tissues and organs differentiate precociously during the larval phase. We have investigated the expression of an adult muscle actin gene during adultation in the ascidian Molgula citrina. Ascidians contain multiple muscle actin genes which are expressed in the larva, the adult, or during both phases of the life cycle. In ascidian species with conventional larval development, the larval mesenchyme cells, which are believed to be progenitors of the adult mesoderm, remain undifferentiated and do not express the muscle actin genes. In M. citrina, the mesenchyme cells differentiate precociously during larval development, suggesting a role in adultation. An adult muscle actin gene from M. citrina was obtained by screening a mantle cDNA library with a probe containing the coding region of SpMA1, a Styela plicata adult muscle actin gene. The screen yielded a cDNA clone designated McMA1, which contained virtually the complete coding and 3' noncoding regions of a muscle actin gene. The deduced McMA1 and SpMA1 proteins exhibit 97% identity in amino acid sequence and may be encoded by homologous genes. The McMA1 gene is expressed in juveniles and adults, but not in larval tail muscle cells, suggesting that it is an adult muscle actin gene. In situ hybridization with a 3' non-coding region probe was used to determine whether the McMA1 gene is expressed during adultation in M. citrina. McMA1 mRNA was first detected exclusively in the mesenchyme cells during the late tailbud stage and continued to accumulate in these cells during their migration into the future body cavity and heart primordium in the hatched larva. The McMA1 transcripts persisted in mesenchyme cells after larval metamorphosis. It is concluded that an adult muscle actin gene shows a heterochronic shift of expression into the larval phase during adultation in M. citrina.