Structural relationships between cardiac isomyosins were analyzed in 10 species using native-gel electrophoresis and radioimmunoassay. In the rat and rabbit, three types of ventricular isomyosin, V1, V2, and V3, were identified by electrophoresis. Monoclonal antibodies specific for the heavy chains of either type V1 or type V3 isomyosin in the rat and rabbit were used for comparison of immunological relationships between atrial and ventricular myosins in other species. Normal guinea pig ventricular myosin reacted with both anti-V2 and anti-V3 antibodies, but only a single myosin band was detected in this species by electrophoresis. When thyrotoxic cardiac hypertrophy was induced in guinea pigs, there was a decrease in myosin reactivity with the anti-V3 antibody and an increase in anti-V1 reactivity. This change in immunological reactivity indicated a change in proportions of two cardiac isomyosins in the guinea pig ventricle even though no myosin heterogeneity was detected by electrophoresis. In six other species including Xenopus, chicken, dog, pig, beef, and human, only a single band of myosin was detected by electrophoresis, and each myosin reacted only with the anti-V3 antibody. In the mouse, three types of ventricular myosin were also detected by electrophoresis. However, unlike V1 isomyosin of the rat and rabbit, mouse V1 isomyosin reacted equally with both anti-V1 and anti-V3 antibodies. In conclusion, we have identified highly conserved epitopes in cardiac myosin, which were found to specifically occur on either the high Ca2+-ATPase type V1 isomyosin or the lower ATPase type V3 ventricular isomyosin in most of the species examined.