Human thymus-leukemia-associated antigen (HThy-L), a saline-soluble antigen, was previously detected by immunodiffusion (but not on the cell surface) in significant quantity in extracts of normal thymocytes, cells from cultured T-cell lines, and erythrocyte-rosette-positive leukemia blasts. Two species of HThy-L were identified and isolated from normal human thymus tissue after extraction in tris buffer, ammonium sulfate fractionation, acid precipitation of inactive fractions, DEAE-cellulose (DE-52) chromatography, Sephadex G-100 gel filtration, and carboxymethyl-cellulose (CM-52) chromatography; On Sephadex G-100, both HThy-L species had a similar molecular weight (40,000--50,000), but they eluted in different positions on DE-52 and CM-52. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that each of the 2 HThy-L species contained 2 components with molecular weights of approximately 43,000 and 23,000. Further purification of HThy-L on Sephadex G-50 showed that the 43,000-dalton component possessed HThy-L activity.