Stimulation of DNA and protein synthesis in brown preadipocytes by 1 microM neokyotorphin in serum-containing media was comparable with the effect of 1 microM norepinephrine. In serum-free medium a decrease and a shift of the maximal effect to lower concentration of neokyotorphin were observed. Kyotorphin had no effect on cell proliferation in either medium; however, 0.01-1 microM kyotorphin inhibited the cell proliferation stimulated by 1 microM norepinephrine. Norepinephrine and both peptides stimulated comparable Ca2+ rise in freshly isolated brown preadipocytes. The effects of neokyotorphin and norepinephrine were additive, whereas 0.03-0.3 microM kyotorphin blocked the action of 3 microM norepinephrine. The peptides did not affect the cAMP level in non-stimulated or norepinephrine-stimulated cultured cells. The effects of the peptides on the brown fat cell cultures indicate that peripheral tissue cells contain receptors for these neuropeptides.