Regulation of the growth and production of granulocyte colony stimulating activity (CSA) by WEHI-3 cells, a lysozyme secreting mouse cell line adapted to culture, was investigated in vitro. WEHI-3 cloning efficiency is not enhanced by exogenously added CSA. WEHI-3 cloning efficiency in agar was suppressed by activity in human polymorphonuclear neutrophil extract (colony inhibiting activity [CIA]) which inhibits endogenous WEHI-3 CSA production. The addition of increasing concentrations of WEHI-3- or L cell [fibroblast]-conditioned medium containing CSA to CIA depressed WEHI-1 agar cultures resulted in graded increases of cloning efficiency to that of the untreated sample. Testosterone, Deca-Durabolin and bacterial lipopolysaccharide increased production of CSA by WEHI-3 cells and overcome CIA mediated suppression of CSA production, even when activating agents were added 1 days after the addition of CIA. The activating agents had no direct stimulatory effect on normal mouse marrow colony forming cells and did not enhance CSA activity. WEHI-3 cells respond to growth inhibitory and stimulatory activities and can serve as an in vitro model for studying the regulation of neoplastic cells.