To investigate the possible role of calmodulin in the calcium sensitivity of parathyroid adenylate cyclase (AC), the effect of the calmodulin inhibitor trifluoperazine hydrochloride (TFP) on the calcium sensitivity of forskolin-stimulated AC activity was investigated in membranes prepared from normal porcine parathyroid glands. TFP inhibited AC in a concentration-dependent manner, the IC50 being approximately 100 microM. The inhibition of the enzyme occurred at roughly the same concentration of TFP in the presence and absence of calcium. Another calmodulin inhibitor, N-(6-aminohexyl)-chloro-1-naphthalenesulfonamide (W-7), also inhibited AC in a calcium-independent manner with a IC50 of approximately 200 microM. The pattern of calcium inhibition of AC was compared in membranes prewashed with either EGTA or 2 microM ionic calcium plus 100 microM TFP in an attempt to remove endogenous calmodulin. Neither treatment significantly altered the apparent affinities of the two previously reported calcium inhibition sites, nor did they alter the relative contribution of the individual calcium inhibition sites to the overall calcium inhibition. Inclusion of 100 microM TFP in the incubation mixture resulted in no change in the apparent affinities of the calcium inhibition site although it did result in a significant decrease in the relative contribution of the high affinity site (P less than 0.05). Addition of exogenous calmodulin (5-50 micrograms/ml) had no significant effect on AC. We conclude from these studies that the inhibition of parathyroid AC by calcium is independent of calmodulin and that this enzyme has intrinsic high sensitivity to calcium.