Acrosome Stabilizing Factor (ASF) has been previously demonstrated to reversibly decapacitate sperm, presumably through preventing the acrosome reaction. ASF is a 259,000 Mr glycoprotein composed of two dissimilar subunits and is synthesized by the corpus epididymis. This report takes advantage of monoclonal antibodies directed toward different antigenic determinants of the ASF macromolecule to develop an immunoradiometric assay. Because the immunoradiometric assay sandwiches the native antigen between two antibodies (one iodinated), this type of assay circumvented the denaturation of ASF caused by iodination. Conditions established for maximal sensitivity with reasonable efficiency were determined to be a 12-24-h equilibrium incubation of ASF with the first bound antibody followed by a carefully timed 3-h incubation with the iodinated second antibody. These conditions provide an assay for ASF that is sensitive to 200 pg/ml and covers a concentration range of more than 2.5 orders of magnitude. The ejaculates from nine male rabbits were evaluated semiweekly over a two-month period for ASF concentration, volume, and sperm number. The average ASF concentration was 265 micrograms/ml of ejaculate and while relatively consistent for any one buck, varied widely between bucks. This variation may relate to the previously reported differences of capacitation of sperm from different bucks.