The effect of vanadate on the adenylate cyclase activity of rat cerebral cortex homogenates is described. In the absence of ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA), 10(-6)M vanadate inhibited enzyme activity by 23%, while 10 (-4) M and 10(-3) M stimulated the enzyme by 14 and 90%, respectively. In the presence of 0.2 mM EGTA, 10 (-6) M to 10(-3) M vanadate had only stimulating effects (18-450%). Additive effects of vanadate and noradrenaline on adenylate cyclase activity suggest different sites of action of these agents. Interaction of vanadate with both fluoride and guanyl-5'-yl imidodiphosphate had an apparently competitive character. Adenylate cyclase maximally stimulated by fluoride (10 mM) was inhibited by vanadate. This inhibitory effect was more pronounced in the absence of EGTA. Adenylate cyclase in the homogenates from the rat cerebral cortex in vivo invaded by spreading depression was slightly increased (up to 38%). This effect was abolished by low (10 (-7) M) vanadate. The results suggest that brain adenylate cyclase is stimulated by vanadate via the guanine nucleotide regulatory protein. The mechanism of vanadate's action, its modulation by calcium ions and the possible physiological role of these effects are discussed.