Previous studies in which an alloantiserum was used to measure Slp levels indicated that in certain inbred strains of mice (Slpa), this protein was sex-limited, that is, present only in males. We raised several monoclonal antibodies directed against different epitopes of Slp and used them to develop a sensitive two-site immunoradiometric assay. Using this assay we detected Slp in serum of all Slpa females previously thought to be phenotypically negative. The levels of Slp in these females are about 0.2 to 4% of that of the males of the same inbred strain. The molecule found in serum of females was isolated by affinity chromatography and was found to have the characteristic three-chain structure of male Slp. These findings establish that the presence of Slp in Slpa females is the rule rather than the exception. Quantitative differences similar in magnitude to those found between males and females were also detected among Slpa males; i.e., Sd males have about 100 times more Slp than Sp males. The mechanisms by which androgens determine the extensive quantitative male-female differences, and by which the S region determines large variations among Slpa males, are unknown.