The effects of VIP receptor antagonists were investigated using non-small cell lung cancer (NSCLC) cells. By Northern blot and RT-PCR, VIP1 receptors were detected on NSCLC cell line NCI-H1299. VIPhybrid,(N-Stearyl-Norleucine17) VIPhybrid ((SN) VIPhybrid) and PTC4495 inhibited 125I-VIP binding to NCI-H1299 cells with IC50 values of 500, 30 and 5000 nM respectively. (SN) VIPhybrid (1 microM) had no effect on basal cAMP but strongly inhibited the increase in cAMP caused by 10 nM VIP. The order of peptide potency to inhibit cAMP was (SN) VIPhybrid > VIPhybrid > PTC4495. (SN) VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 colony formation. Also, (SN) VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 xenograft formation in nude mice. These data suggest that (SN) VIPhybrid antagonizes VIP1 receptors on NSCLC cells.