Under monitoring the antigenic activity by radioimmunoassay (RIA), one of the major sea-squirt antigens was isolated by gel chromatography after treatment of the extract from internal organs of sea-squirt with QAE-Sephadex. This antigen was fairly homogenous as judged from its elution profile in chromatography on a column of Ultrogel AcA 44 with respect not only of the antigenic activity but also to UV absorption. Furthermore, the antigen inhibited the binding of the 125I-labeled tracer antigen to rabbit anti-sea-squirt antibody in a similar mode to the case of Ei-2, one of the sea-squirt antigens previously prepared, but its specific activity was as high as 2.33 times that of Ei-2. Accordingly this isolated antigen was corresponded to the major antigenic substance in Ei-2 and referred to as Ei-M. In addition, the other antigen of Gi-x was also isolated by chromatography on a column of Sepharose 6B as the major antigenic substance in Gi-2, the other one of the previous antigens. Gi-x was apparently different from E-M in the mode of inhibition of the binding of the tracer antigen to the antibody, although its antigenic activity against the asthmatic patients in vivo was as high as that of Ei-M. Thus, it was considered that sea-squirt contained at least two types of antigens of Ei-M and GI-X.