The accuracy of radioimmunoassay (RIA) has been much improved for sea-squirt antigen Gi-x, which is considered to be of higher molecular weight than antigen Ei-M, by using radiolabeled Gi-x as a tracer. Gel chromatography monitored by the improved RIA revealed a wide distribution of molecular weight of Gi-x type antigens, in contrast to Ei-M. However, since a considerable portion of the anti-genic activity gave a single peak in gel chromatography with Sepharose 6B, the substance in the peak fractions was isolated as a fairly homogeneous preparation and referred to as Gi-rep. Gi-rep showed distinct characteristics of Gi-x type antigens and was clearly discriminated from Ei-M by radioimmunometry in vitro. The in vitro observation also suggested that Gi-rep and Ei-M carried a common antigenic determinant (type alpha), but that Ei-M also carried a specific determinant (type beta). The weight-average molecular weights were 1.1 x 10(5) for Gi-rep and 2.3 x 10(4) for Ei-M. Both preparations consisted of acidic glycoproteins with considerable amounts of sulfate and phosphate.