Microtiter immunoassays were used to determine whether a panel of 53 monoclonal antibodies submitted to the ISOBM TD-3 PSA Workshop could form assay combinations with free prostate-specific antigen (PSA) and the PSA complex with alpha1-antichymotrypsin. A panel of 6 known anti-PSA antibodies (H117, H50, H179, H164, 2E9 and 5A10) was used as labelled tracers. Epitope groups were proposed based on the ability of the Workshop antibodies to form good assay combinations with these 6 known anti-PSA antibodies. Nine of the TD-3 Workshop antibodies were found to react only with free PSA. Two additional epitope clusters were identified with 8 antibodies showing similar reactivity to antibody H117, while 11 antibodies formed a different cluster showing similar reactivity to antibody H50. Defining the nature of these immunodominant regions will be valuable in the development of more appropriate immunoassays for PSA.