The ventricles of rabbit heart contain three isomyosins (V1, V2, and V3) whose proportions change during development, and after treatment with thyroid hormone. The V1 isomyosin accumulates rapidly after administration of thyroid hormone and we have shown that this results from marked change in the synthesis rates of the heavy chains of both V1 (HC alpha) and V3 (HC beta). The fractional rates of synthesis were calculated from the specific radioactivities of leucine in plasma and in HC alpha and HC beta after continuous infusion with [3H] leucine for 1 h. The V1 and V3 isomyosins were separated by affinity chromatography with monoclonal antibodies and the heavy chains were then isolated by sodium dodecyl sulfate-electrophoresis. In 32- to 35-day-old euthyroid rabbits, the fractional rates of synthesis of the two heavy chains were not significantly different with a combined mean rate of 0.14 day-1. Administration of 3,5,3',5'-tetraiodothyronine (200 micrograms/kg/day) caused a 3-fold increase in the synthesis rate of HC alpha and a comparable decrease in that of HC beta. The fractional synthesis rate of atrial myosin heavy chain also changed when 3,5,3',5'-tetraiodothyronine was administered, increasing from 0.15 day-1 to an average of 0.20 day-1 after 1 day of treatment. This was not accompanied by any change in the heavy chain type as determined by radioimmunoassay.