This study demonstrates that the inhibition of adenylate cyclase activity by Ca2+ is enhanced in the presence of increasing [EGTA] (0, 0.3, 1, 2.5 mM) by 2 orders of magnitude. It has been established that this effect is not because of poor Ca2+ buffering by low [EGTA] or high Ca2+ binding by the membrane preparation. It is present irrespective of stimulus. We suggest the enhanced sensitivity of adenylate cyclase to Ca2+ induced by EGTA is caused by the Ca-EGTA complex being a more inhibitory species than Ca2+. Thus consideration of the effects of the Ca-EGTA complex should be made when interpreting the results from experiments involving Ca2+ and EGTA.