The high-affinity receptor for melatonin is coupled to a pertussis toxin-sensitive, inhibitory guanine nucleotide regulatory protein, Gi, which mediates inhibition of adenylate cyclase activity in the chick and other species. Heparin has been found to uncouple alpha 2-adrenoceptors from a similar Gi; therefore it was of interest to examine the effect of this agent on melatonin binding and signal transduction in chick brain. In competition studies, melatonin inhibited the binding of 2-[125I]iodo-melatonin with high- and low-affinities of KH = 20 pM and KL = 15 nM, respectively. In the presence of heparin (100 units/ml), a single site with an affinity of about 6 nM was detected. The monovalent cations, Na+ and Li+, produced a greater rightward shift of the agonist competition curve than heparin and converted all high-affinity sites to a low affinity of approximately 15-18 nM. In saturation studies, heparin reduced the affinity of high-affinity sites and caused a significant decrease in the density of low-affinity sites. In keeping with its ability to reduce high-affinity binding, heparin blocked melatonin's inhibitory effect on forskolin-stimulated adenylate cyclase activity in chick brain synaptosomal membranes. These findings indicate that heparin impairs the coupling between the melatonin receptor and Gi, with a consequent decrease in binding affinity and a loss of receptor-mediated signalling.