The effect of melatonin on glucose metabolism in the presence and absence of insulin has been investigated in the primary cultures of renal tubules grown in a defined medium. In the absence of glucose in the medium containing 5 microg/mL of insulin and 2 mm alanine + 5 mm glycerol + 0.5 mm octanoate, 100 nm melatonin stimulated both glucose and lactate synthesis, while in the medium devoid of insulin melatonin action was negligible. Melatonin-induced increase in glucose and lactate synthesis was accompanied by an enhancement of alanine and glycerol consumption. In view of measurements of [U-14C]L-alanine and [U-14C]L-glycerol incorporation into glucose, it is likely that melatonin increased alanine utilization for glucose production, while accelerated lactate synthesis was because of an enhanced glycerol consumption. As (i) 10 nm luzindole attenuated the stimulatory action of melatonin on glucose formation and (ii) the indole induced a decrease in intracellular cAMP level, it seems likely that in renal tubules melatonin binds to ML1 membrane receptor subtype. In view of a decline of intracellular fructose-1,6-bisphosphate content accompanied by a significant rise in hexose-6-phosphate and glucose levels, melatonin might result in an acceleration of flux through fructose-1,6-bisphosphatase probably because of an increase in the active, dephosphorylated form of this enzyme. Thus, the administration of melatonin in combination with insulin might be beneficial for diabetic therapy because of protection against hypoglycemia.