The hemoglobin of the erythrocytes of Urechis caupo is tetrameric. Three chromatographic fractions have been isolated. Fractions F-I (the major fraction) and F-II are each composed of at least five electrophoretic components. Minor fractions F-III has an amino acid composition which is very different from those of fractions F-I and F-II. Amino acid analysis and peptide mapping suggests that fractions F-I and F-II are very similar, and may differ only in a few post-translational modifications. The major fraction, F-I, appears to consist of tetramers in which all subunits are almost identical. Oxygen binding is noncooperative, with a pressure of half-saturation (p50) of 12 mm Hg at 20 degrees C and pH 7.5 in Tris buffers. The oxygen equilibria of undialyzed lysate and of hemoglobin purified by chromatography on Sephadex G-100 are identical. This observation indicates that no allosteric modulator is present. The oxygen equilibrium is not significantly affected by ATP, Cl-, Ca2+, Mg2+, or CO2. The pH dependence of oxygen binding is extremely small: delta Log p50/delta pH is only +0.06 between pH 6 and 7.5. The p50 value changes by no more than 15% between 6.8 microM and 4.3 mM (heme). The apparent enthalpy of oxygenation, delta H, is -11 kcal/mol. Deoxygenation, oxidation, dilution, or pH changes have no significant effect on the state of aggregation. These oxygen-binding properties are consistent with the suggestion that the primary function of the hemoglobin is to store oxygen during the hypoxia which occurs in the burrows of the animal at low tide.