Myosin accumulation was examined in developing ascidian embryos by measuring its ATPase activity and by resolving myosin heavy chains on polyacrylamide gels. Both procedures used a myosin-enriched fraction that was prepared by exploiting the unusual solubility properties of this protein. Myosin ATPase was first observed at neurulation and increased 25to 50-fold by the time of larval hatching, a sequence similar to that found previously for muscle acetylcholinesterase. This similarity to acetylcholinesterase, and the finding of at least two-thirds of the ATPase activity in the larval tail, imply that most of the myosin studied was muscle myosin. The pattern of expression based on ATPase assays was confirmed in part by gel analysis, but since this technique was less sensitive the appearance of myosin heavy chain was not demonstrated unambiguously until later in development. As found earlier with acetylcholinesterase, actinomycin D treatment only interfered with accumulation of myosin ATPase during early stages of increasing enzymatic activity. Despite certain similarities noted in their ontogeny, differences in the initial increase in the activity of these enzymes and the time of their first response to actinomycin D suggest that the events concerned with the expression of acetylcholinesterase occur 1 hour before those of myosin ATPase. The appearance of these two proteins, representing different enzymes of the muscle cell, is probably controlled independently.