This study investigated the hypothesis that changes in the adenine nucleotide (ATP + ADP + AMP) content of kidney mitochondria can occur by a transport mechanism that catalyzes net transfer of adenine nucleotides across the inner mitochondrial membrane. The adenine nucleotide content of isolated kidney mitochondria was 8.23 +/- 0.85 nmol/mg mitochondrial protein. This amount increased or decreased as a function of the external [ATP-Mg] when mitochondria were incubated in phosphate-containing medium. The increases and decreases were inhibited to different extents by 100 microM EGTA (ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid) or 5 microM carboxyatractyloside (CAT), suggesting two transport mechanisms. The unidirectional components (influx and efflux) of net flux were examined separately for the CAT-insensitive (EGTA-sensitive) and CAT-sensitive (EGTA-insensitive) mechanisms. CAT-insensitive adenine nucleotide influx and efflux were stimulated by [Ca2+]free up to 2 microM; for ATP influx, Km was 1.7 mM, Vmax was 3.5 nmol/min/mg protein, and Mg2+ was required. Efflux varied as a function of both the external and matrix [ATP] and was completely inhibited by mersalyl. ATP was a better substrate than ADP, and ADP transport did not require Mg2+. The CAT-sensitive mechanism was characterized by studying phosphate-induced adenine nucleotide efflux. Efflux varied with external [Pi] and with matrix [ATP] and was not inhibited by cyclosporin. The amount of CAT required for maximal inhibition was 800 pmol/mg protein. In contrast to CAT-insensitive efflux, this pathway was only partially inhibited by mersalyl and showed no preference for ATP vs ADP. In conclusion, two distinct mechanisms for net adenine nucleotide transport were demonstrated. Both exchange adenine nucleotides (ATP-Mg or ADP) for Pi. One mechanism is identical to the CAT-insensitive ATP-Mg/Pi carrier known in liver mitochondria; the other is a CAT-sensitive mechanism that is not present in liver and may represent a novel function of the ADP/ATP translocase or another CAT-sensitive carrier.