From the extracellular hemoglobin of Amphitrite ornata four constituent polypeptide chains containing heme and designated AI, AII, BI and BII according to the elution order were obtained by DE52-cellulose ion-exchange chromatography with dithiothreitol (DTT) as a reducing reagent. The NH2-terminal sequences for the chains are AI, Asp-Ser-Asn-Ala; AII, Glu-Tyr-Thr; BI, Asp-Phe-Asn-Thr; and BII, Asp-Ser-Glu. Each of the isolated chains showed spectra similar to those of vertebrate hemoglobins, and they bound oxygen reversibly. Acid urea polyacrylamide gel electrophoresis separated four bands, corresponding to the isolated chain, from the intact extracellular hemoglobin reduced with DTT. These results and our failure to detect an appreciable amount of non-heme protein suggest that the extracellular hemoglobin of A. ornata is composed of four polypeptide chains, each containing a heme.