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Polyacrylamide and starch gel electrophoresis of the hemoglobin of the killifish Fundulus heteroclitus reveal the presence of four clearly distinguishable components. These isohemoglobins, each tetramers consisting of alpha and beta chains, can be preparatively separated by ion exchange chromatography on DEAE-cellulose and are homogeneous according to isoelectric focusing in polyacrylamide gels. Oxygen equilibria of the isolated hemoglobin components (Hb I, Hb II, Hb III, and Hb IV) show only minor differences in the magnitude of the Bohr effect and in the effect of ATP on the binding of oxygen. Four different globin chains, alphaa, alphab, betaa, and betab, can be separated by ion exchange on CM-cellulose. Hb I is a homotetramer of alphab and betab chains, Hb IV consists of alphaa and betaa subunits, and components II and III are heterotetramers consisting of all four chains. The alpha and beta chains differ significantly in amino acid composition. A model suggesting the existence of 10 different isohemoglobins, 6 of which have stable intersubunit contacts, has been proposed to account for the qualitative and quantitative aspects of the electrophoretic behavior of the components. Separations of the isohemoglobins on DEAE-cellulose under slightly modified conditions provide additional support for the model.