hCG is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and is normally synthesized by trophoblastic tissue. Its measurement by immunoassay is widely employed as a test for pregnancy, but can be complicated by cross-reactivity with human (h) LH. Immunoassays based on the beta-subunit of hCG have been employed to decrease this cross-reactivity with hLH, but when these assays are used with urine specimens, the antibodies employed also detect a fragment of hCG beta, which can lead to significant differences in measurement. To overcome these problems, we have developed a series of monoclonal antibodies to the beta fragment of hCG recovered from pregnancy urine. Some of the antibodies that bind to this beta fragment are directed to a region of hCG beta that is different from the epitopes recognized by antibodies raised against the intact beta-subunit. The new epitopes available in the hCG beta fragment form the basis for novel immunoassays. These beta fragment antibodies are used in conjunction with other antibodies, directed to different epitopes of the hormone, to produce a series of immunoradiometric assays that can discriminate among intact hormone, free hCG beta, and hCG beta fragment. The hCG beta fragment antibodies described herein have affinities between 10(9) and 10(11) M-1 for the beta fragment and exhibit varying degrees of discrimination between the hCG beta fragment, the beta-subunits of hCG and hLH, and intact hCG and hLH.