The effects of bombesin (BB) on mitogen activated protein (MAP) kinase were investigated using non-small cell lung cancer (NSCLC) cells. By Western blot, both 42 and 44 kDalton forms of MAP kinase were present in NCI-H1299 and NCI-H838 cells. Addition of BB to NCI-H1299 cells resulted in phosphorylation of the MAP kinase substrate myelin basic protein (MBP). Phosphorylation of MBP was maximal 6 min after the addition of 10 nM BB to NCI-H1299 cells. Addition of gastrin releasing peptide (GRP) or GRP14-27 but not GRP1-16 to NCI-H 1299 cells caused MBP phosphorylation. The effects of BB were inhibited by BW2258U89, a BB receptor antagonist, and PD98059, a MAP kinase kinase inhibitor. Also, PD98059 inhibited the clonal growth of NCI-H1299 cells. These data suggest that MAP kinase may be an important regulatory enzyme in NSCLC.