Culture supernatants from concanavalin A activated murine splenic mononuclear cells (splenocytes) were assayed for prolactin-like molecules using the Nb2 node lymphoma bioassay and immunostaining procedures. Con A stimulated splenocytes produced a factor with prolactin-like immunoreactivity that was mitogenic for Nb2 lymphoma cells. This factor first appeared in culture supernatants with the onset of lymphocyte mitogenesis and its activity was proportional to the number of cells cultured. In addition, splenocyte proliferation was inhibited by antiserum to rat prolactin, and this inhibition was partially reversed by added hormone. These findings show that murine splenocytes produce a substance with prolactin-like activity and suggest that this factor is essential for lymphocyte proliferation.