A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD(+) at 25 degrees C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl(2), and 5 millimolar alpha-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD(+) efflux was observed instead. Furthermore, the rate of NAD(+) accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3'-NAD(+). When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD(+) content. This led to a slow decrease of NAD(+)-linked substrates oxidation by isolated mitochondria The rate of NAD(+) efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD(+) transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.