Bombesin-like peptides including neuromedin B have been proposed as autocrine or paracrine growth factors for carcinomas. To examine signal transduction and regulation of cell growth by NMB, transfectants were created with the rat NMB receptor (NMB-R) gene in BALB/3T3 cells which do not express an endogenous bombesin peptide receptor. The resultant cell line, NMB-8, expresses 800,000 NMB binding sites/cell. Addition of NMB has a biphasic effect on [3H]thymidine ([3H]dT) incorporation in confluent and quiescent cells: up to 10 nM of NMB causes a 1.5-3-fold stimulation of [3H]dT incorporation, but at greater than 10 nM there is inhibition of [3H]dT incorporation, and at 100 nM of NMB there is inhibition of cell growth. NMB causes protracted increases in intracellular Ca2+, and pertussis toxin (PT)-insensitive phosphatidylinositol (PI) turnover. NMB-mediated increase in membrane phospholipase-C activity is stimulated by guanosine 5'-O-(3-thiotriphosphate). Arachidonate release is also activated by NMB in a PT-insensitive manner. Brief exposure to 12-tetradecanoylphorbol 13-acetate inhibits NMB-mediated PI turnover but not arachidonate release. Thus, in NMB-8 cells, distinct mechanisms govern NMB-mediated phospholipase-C activation and arachidonate release. Also, neuromedin-B is potentially a bifunctional regulator of cell growth.