The adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of crude Chinese hamster ovary cell membranes was inhibited 30-40% by low concentrations (6-600 ng/ml) of calcium-dependent regulator (CDR). This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The adenylate cyclase activity of membranes prepared from low population density Chinese hamster ovary cells was not appreciably altered by CDR. However, with increasing cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. Further, the intracellular levels of CDR determined in the 12,000 x g supernatant and particulate fractions varied inversely with increasing cell population density. As cell number increased from 2 x 10(6) to 10 x 10(6) cells per dish the CDR concentration present in the supernatant fraction increased from 0.4 to 0.8 mug of CDR per mg of protein, while the amount of endogenous CDR associated with the particulate fraction decreased from 0.6 to 0.4 mug of CDR per mg of protein. This suggests that possible changes in the distribution of CDR between the supernatant and membrane fractions might serve as a regulatory mechanism for activities under CDR control.