We have previously used measurements of uncoupler-enforced reverse activity to demonstrate that the mitochondrial Ca2+ uniporter is strongly inhibited by external EGTA plus free Mg2+, following a brief period of rapid activity. Using the same approach, we now show that in addition to divalent cations, the uniporter is regulated by external adenine nucleotides and by other components of the cytosol. Inhibition produced by EGTA plus free Mg2+ is reversed by spermine (EC0.5 approximately 40 microM) and reduced when mitochondria are purified by an isoosmotic density-gradient method. Under either condition, inhibition is restored by external adenine nucleotides in a concentration-dependent manner. The order of effectiveness is ATP > ADP > AMP, with the nucleoside adenosine being ineffective. Among nucleotide triphosphates, the order is ATP > CTP approximately UTP > GTP. The effectiveness of ATP (EC50 approximately 0.6 mM) is the same in mitochondria and mitoplasts, the same as that of AMPPNP, and is not altered by the presence of oligomycin, carboxyatractyloside, or AP5A, used alone or in combinations. These findings indicate that ATP acts at a site located on the outer surface of the inner membrane through a mechanism which does not require its hydrolysis. Phosphate also inhibits reverse uniport under some conditions (EC50 approximately 20 microM). The sites at which free ATP and free Mg2+ inhibit the uniporter can be distinguished by chymotrypsin treatment of mitoplasts, which eliminates the action of Mg2+ but does not affect the action of ATP. Data are interpreted within the context of a model in which the uniporter is considered to be a gated channel that is controlled, in part, by specific external effector sites that accept divalent cations or nucleotides. The possible consequences of the model for cell Ca2+ regulation by mitochondria and regulation of TCA cycle activity by the matrix free Ca2+ concentration are considered.