A competitive radioimmunoprecipitation method was developed for the quantitation of human thymus-leukemia-associated antigen (HThy-L) isolated from human thymus tissue. Antisera to the antigen were raised by immunization of rabbits with purified fractions of HThy-L. The antigen was labeled with 125l by means of a modification of the lactoperoxidase technique and subjected to Sephadex G-100 gel filtration. Analysis of fractions eluted from this column by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two labeled components with apparent molecular weights of 45,000 and 25,000 daltons, respectively. Immunoprecipitation with anti-HThy-L antiserum demonstrated directly that the 45,000-dalton component carried HThy-L antigenicity. This fraction served as the source of labeled antigen in a competitive radioimmunoassay that permitted the detection of approximately 1 ng HThy-L. With the use of this assay, we confirmed quantitatively that the highest amounts of HThy-L were found in extracts of thymocytes, normal thymus tissues, and lymphoblasts for T-cell lines.