In this study, immunochemical techniques were employed to examine the guinea pig (GP) testicular proacrosin-acrosin system. Monospecific polyclonal antibodies to the highly stable enzymatically active 34,000 molecular weight form of GP testicular acrosin were generated. Western blot analysis of acid extracts prepared from snap-frozen freshly excised GP testes revealed two major immunoreactive bands with mol. wts of approximately 62,000 and 48,000 and one minor band with an approximate mol. wt of 54-56,000. The 62,000 mol. wt molecule identified is in close agreement with the previously reported mol. wt for purified GP testicular proacrosin. Western blot analysis of different species of testicular acid extracts demonstrated the evolutionary relatedness of the proacrosin-acrosin systems since immunoreactivity was observed primarily with acid extracts from rodent species (rat, mouse and hamster) and not with extracts from evolutionarily less-related species (goat, ram and bovine). The majority of the cross-reactivity observed was characterized by immunoreactivity with the 62,000 and 48,000 mol wt molecules. The only species which exhibited cross-reactivity with the 54-56,000 mol. wt protein was rat. In addition, the iso-immunogenic and aspermatogenic properties of the 34,000 mol. wt form of GP testicular acrosin were examined. One out of five Hartley and one out of seven Strain 2 female GPs immunized and boosted with a total of 200 micrograms of purified protein exhibited increased levels of circulating anti-acrosin iso-antibodies. The antigenic specificity of the iso-antibodies observed in the two animals was verified by Western blot analysis. All other female animals, including two strain 13 GPs, exhibited very low or undetectable levels of such antibodies following immunization. One out of three male Hartley GPs immunized with 50 micrograms of the purified protein exhibited typical lesions of experimental allergic orchitis while none of a group of three animals developed lesions at a 5 micrograms dose. Taken together, these results suggest that the 34,000 mol. wt form of GP testicular acrosin is neither a highly potent iso-immunogen nor aspermatogenic autoantigen.