Seven mouse monoclonal antibodies (IgGs) were produced against rat neurophysins (NPs). Three were specifically directed against vasopressin-associated NP (NP-AVP), and four were specific for oxytocin-associated NP (NP-OT). These specificities were observed in liquid phase assays, immunoblot, and immunoprecipitation experiments. Homozygous Brattleboro rat tissues and extracts, which do not contain vasopressin or NP-AVP, did not react with the anti-NP-AVP antibodies but reacted with high affinity to the anti-NP-OT antibodies. In immunoprecipitation assays the antibodies brought down the appropriate NPs as well as their precursor molecules synthesized in vivo with no detectable cross-reactivity. In solid phase assays where the antigens were presented in a different manner, there was a significant cross-reactivity of the anti-NP-AVP antibodies with NP-OT. The extent of this cross-reactivity in solid phase correlated with the cross-reactivities of the antibodies observed in immunocytochemical studies. These solid phase (and immunocytochemical) data demonstrated that liquid phase specificities and absorption controls of antibodies are inadequate to assess their immunocytochemical (solid phase) specificities. Posterior pituitary extracts from the mouse and frog, as well as purified NPs from the rat, cow, and human were studied for their cross-reactivities to two of the antibodies, PS 36 and PS 45. In liquid phase assays the anti-rat NP-OT antibody, PS 36, reacted only with rat and mouse NPs and did not cross-react with NPs from any of the other species. In contrast, the anti-rat NP-AVP antibody, PS 45, was cross-reactive across species lines including an NP-like antigen extracted from frog posterior pituitaries. Immunoblot staining with these antibodies showed heterogeneity of NP-AVP and NP-OT in the rat posterior pituitary. Analysis of the epitopes for PS 36 and PS 45 indicated the antigenic determinants were located near amino acid positions 80 to 81 in NP-OT and 75 to 86 in NP-AVP, respectively.