Rat adrenal mitochondria contain approximately equal levels of P450 scc and P450 11β, each reduced by NADPH through adrenodoxin reductase (ADX-reductase) and adrenodoxin (ADX). Constitutive cholesterol side-chain cleavage (SCO can be increased over 20-fold through a combination of hormonal activation and inhibition of cholesterol metabolism in vivo prior to isolation of the mitochondria. This stimulation, which results from accumulated reactive cholesterol, does not significantly affect either the dependence of activities on the concentration of isocitrate (IC) and succinate (SU) or the ratio of maximum activities [3:1]supported by these reductants. Thus, the rate of cholesterol SCC is determined independently by electron transfer and the amount of reactive cholesterol. Hydroxylation of deoxycorticosterone (11β and 18 positions) required much higher levels of each reductant, indicating less effective reductant transfer to P450 11β. Reactions at P450 scc and P450 11β, mediated by IC, are enhanced by low concentrations of various dicarboxylates anions (fumarate, SU). The actions of SU dehydrogenase inhibitors and the activity of fumarate, a poor direct reductant, suggest that higher production of NADPH results from malate-enhanced uptake of isocitrate. Only synergistic combinations of reductants are sufficient to sustain maximum rates of 11-deoxycorticosterone (DOC) metabolism, whereas IC is fully effective for P450 scc. Increased reaction at P450 scc (cholesterol loading or addition of 20α-hydroxycholesterol) decreased simultaneous DOC metabolism at P450 11β in inverse proportion to the estimated intramitochondrial generation of NADPH (1 mM or 10 mM SU > 1 mM IC > 10 mM IC). These decreases were reversed by inhibition of P450 scc. Crossover inhibition caused by maximum DOC metabolism was less pronounced. EGTA/albumin treatment, which enhanced activities at both P450 scc and P450 11β, presumably via increased NADPH, diminished this cross-competition. The differential dependence on reductants and the characteristics of crossover competition are consistent with a roughly three-fold more favorable partitioning of electron transfer to P450 scc, possibly caused by preferential interaction of reduced adrenodoxin with P450 scc.