The pineal hormone melatonin modulates constitutive protein secretion from murine melanoma M2R cells in vitro, in a cholera-toxin (CTX)-sensitive process, without effecting major changes in cAMP. The effects of melatonin on GTP binding proteins and putative CTX substrates in these cells were investigated. Melatonin enhanced GTP gamma 35S binding and the incorporation of 32P-P3-(4-azidoanilido)-P1-5'-guanosine triphosphate (Az-32P-GTP) into 94, 40 and 28 kilodalton proteins. Similar changes were induced by CTX treatment. In addition, melatonin enhanced ADP ribosylation of several proteins, among them 94 and 40 kilodalton bands, apparently at arginyl residues. CTX catalyzed the ADP ribosylation of 45 and 40 (both recognized by antibodies specific to the C-terminal peptide of the Gs alpha subunit) and 94 kilodalton proteins and attenuated melatonin's effect. The melatonin-mediated ADP ribosylation reactions were attenuated by nicotinamide which inhibits mono(ADP ribosyl)transferases and poly(ADP-ribose)synthetase, but not by 3-amino benzamide, a specific inhibitor of poly(ADP-ribose)synthetase. Nicotinamide but not 3-amino benzamide prevented the enhancement by melatonin of GTP gamma 35S binding. These results indicate that melatonin enhances protein ADP ribosylation and consequently GTP exchange in a number of CTX-sensitive G proteins. They demonstrate a novel route for concerted activation of multiple GTP binding proteins by a single hormone.