Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10 −4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10 −2 M). Isoporterenol (10 −4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E 1 (10 −5 M) produced similar responses in the two strains. G i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G i2α and G qα/G 11α were similar in the two strains, while the levels of G sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ∼20% in MHS membranes. The α-subunit of G i3 was dramatically reduced by ∼80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.