In Oligobrachia mashikoi, a mouthless and gutless polychaete known as a beard worm, sites of production of extra-cellular giant hemoglobin were examined with whole-mount in-situ hybridization and semi-quantitative RT-PCR. An RNA probe was prepared from mRNA of the A2-globin subunit. Clear signals were obtained from a peritoneal membrane covering the trophosome in the posterior body in all seven individuals examined in this study. In addition, weak signals were observed in the peritoneal membrane covering tissues in the middle part of the body in some individuals. Furthermore, in one individual, signals were obtained in complicated bodies invaginated into the dorsal vessel from a peritoneal membrane that also released signals. The results of RT-PCR regarding the expression levels of four kinds of globin-subunit genes suggest that the main site of hemoglobin production is the peritoneal membrane in the posterior body.