We have described the expression of the prolactin (PRL) gene and the occurrence of PRL-like immunoreactive proteins in the hypothalamic-neurophypophyseal system of the rat. Here, we investigated the nature of neurohypophyseal PRL-like antigens, by studying the biological activity of medium conditioned by incubated neurohypophyses in specific bioassays for PRL and for the 16 kDa N-terminal fragment of PRL (16K PRL). Neurohypophyseal conditioned medium (NHCM) obtained after incubating neurohyphyseal lobes (1 h at 37°C) was enriched with proteins of 14 kDa and 23 kDa, that crossreacted with PRL-and 16K PRL-directed antisera. The NHCM stimulated in a dose-dependent fashion the proliferation of Nb2-lymphoma PRL-dependent cells. This effect paralleled that of PRL and 16K PRL standards and was neutralized by different dilutions of both PRL-and 16K PRL-antisera. Also, the NHCM inhibited the proliferation of endothelial cells in culture, an antiangiogenic-effect exerted by 16K PRL. The antiangiogenic effect of the NHCM was parallel to that of 16K PRL standard and neutralized by 16K PRL antiserum in a dose-dependent fashion. These results indicate that NHCM contains proteins that share receptor activation properties as well as antigenic determinants with both PRL and 16K PRL.