The stimulation of adenylate cyclase activity by guanine nucleotides, isoproterenol and fluoride has been investigated in KC1 washed rat aortic membranes. We observed that the presence of guanine nucleotides in the assay was obligatory for the isoproterenol stimulation of adenylate cyclase activity. Guanine nucleotides could stimulate adenylate cyclase in the absence of catecholamines; however, the extent of stimulation was significantly lower (p < 0.05) than that observed with isoproterenol. The order of effectiveness of guanine nucleotides was guanosine 5’-0-(3-thiotriphosphate) (GTP-γ-S), 5’-guanylylimido diphosphate (Gpp(NH)p), and guanosine 5’-triphosphate (GTP). GTP stimulated enzyme activity without a noticeable lag period, while stimulation by Gpp(NH)p showed a lag period of 3–4 min. Addition of isoproterenol neither abolished the lag period, nor altered the apparent K<sub>a</sub> value (concentration required for half maximal stimulation) for Gpp(NH)p stimulation. In a comparative study of adenylate cyclase activity between spontaneously hypertensive rats (SHR) and Kyoto Wistar normotensive rats (WKY), using 0.6 M KC1 washed membranes from aorta and caudal artery, no differences were observed in the basal adenylate cyclase activity. However, guanine nucleotide-, isoproterenol-, and fluoride-stimulated enzyme activity was significantly lower (p < 0.05) in the caudal artery of SHR as compared to WKY.