Several aspects of the activation of adenylate cyclase by guanosine 5'-triphosphate (GTP), 5'-guanylylimidodiphosphate (Gpp(NH)p) and bovine parathyroid hormone (bPTH) have been studied in chick kidney plasma membrane preparations. GTP (10(-4) mol/l), Gpp(NH)p (10(-4) mol/l) and bPTH (10 i.u./ml) activated adenylate cyclase without any significant time lag. However a 2 min delay was observed before the activity of the enzyme increased after the addition of bPTH (-6 leads to +34) to incubations. The early (0-3 min) effects of GTP and Gpp(NH)p upon chick kidney adenylate cyclase activity were antagonized by the addition of the alternative guanyl nucleotide. After 5 min of incubation with kidney plasma membranes, Gpp(NH)p induced a stable state of activation of adenylate cyclase which was not reversible by subsequent addition of GTP. GTP did not induce an irreversible state of enzyme activation. In pre-incubation studies, GTP did not produce a persistent enzyme activation and did not modify the effect of Gpp(NH)p added subsequently at the incubation stage. Gpp(NH)p produced a stable state of activation of adenylate cyclase which was not inhibited by addition of GTP at the incubation stage. Bovine PTH (2-34) inhibited the effect of bPTH upon adenylate cyclase activity when the native hormone (10 i.u./ml) had been incubated with plasma membranes for up to 8 min before addition of the analogue (5 mug/ml). Incubation of plasma membranes with bPTH (2-34) for as little as 10 s prevented activation of adenylate cyclase by subsequent addition of bPTH. This pattern was confirmed in pre-incubation studies. After pre-incubation of kidney membranes with bPTH and bPTH (2-34), followed by washing, an acid extract of the membranes contained immunoreactive bPTH. Gpp(NH)p produced a greater increase in adenylate cyclase activity in membranes pre-incubated with bPTH or bPTH (2-34) than in membranes pre-incubated with buffer alone, suggesting that the hormone and analogue facilitated the interaction of Gpp(NH)p with adenylate cyclase.