OBJECTIVE The human tissue kallikrein family contains three closely related proteases: human kallikrein 1 (hK1), human kallikrein 2 (hK2), and prostate-specific antigen (PSA). The structural homology between these three proteins suggests potential cross-reactivity interference when different immunologic techniques are used. This study evaluated PSA and hK2 monoclonal antibody (mAb) and polyclonal antibody (pAb) reactivities to hK1, hK2, and PSA. METHODS mAbs and pAbs to hK2 and PSA were evaluated using Western blot analysis on hK1, hK2, PSA, and seminal plasma. RESULTS pAbs to PSA and hK2 recognized all three human kallikreins, as well as fragments of hK2 and PSA. An mAb with minimal (less than 0.4%) cross-reactivity between PSA and hK2 and a cross-reactive mAb were found. mAbs specific to PSA or hK2 did not cross-react with the less homologous hK1 protein. A PSA mAb raised specifically to PSA fragments recognized both PSA and hK2 but did not cross-react with hK1. pAbs to hK1 cross-reacted slightly with PSA and not at all with hK2. CONCLUSIONS Both pAbs and mAbs to hK2 and PSA may exhibit immunocross-reactivity. pAbs to PSA or hK2 react with all three human tissue kallikreins. The potential for cross-reactivity should be considered in any clinical or research procedures that use hK1, hK2, and PSA antibodies.