Guanyl nucleotide regulation of parathyroid hormone (PTH)-activated adenylate cyclase was studied in membrane preparations of cultured opossum kidney cells. Guanosine triphosphate (GTP) (100 microM) decreased PTH-stimulated activity by 70%. Pertussis toxin enhanced PTH stimulation in intact cells and membranes, completely blocked the inhibitory effect of GTP, and catalyzed the [32P]ADP-ribosylation of a 38,000-dalton protein migrating in the position of the alpha-subunit of the inhibitory GTP-regulatory protein Ni. Cholera toxin was used to identify the alpha-subunit of the stimulatory GTP-binding protein Ns, a 42,000-dalton protein. We tested the idea that Ni may be involved in mediating the reduced response of opossum kidney cells to PTH after pretreatment with the hormone (desensitization). GTP inhibited PTH-stimulated activity to approximately the same degree in membranes from PTH-pretreated cells and control cells whether or not the cells had also received pertussis toxin. We conclude that GTP inhibits PTH action in opossum kidney cells through Ni but that PTH-induced desensitization is not mediated by Ni.