Titrations of mitochondrial apo-aspartate aminotransferase with pyridoxal 5'-phosphate in the presence of AMP, contrary to what has been observed in the case of the cytosolic isoenzyme [(1983) FEBS Lett. 153, 98-102], show sigmoidal isotherms, with Hill coefficients ranging from n H = 1.4, in the absence of AMP, to n H = 1.8, in the presence of 5.9 mM AMP. The experimental data were successfully fitted by the Monod-Wyman-Changeaux model. The best fit, in the absence of AMP, was obtained with L = 30, K R = 4.72 x 10 - 7 M and K T = 1.18 x 10 - 5 M. Binding curves in the presence of AMP fit the model by keeping K R as a constant. This implies that AMP could bind to the apoenzyme only in the T state. In contrast, binding curves in the presence of phosphate ion (P i ) showed a less pronounced cooperativity, the Hill coefficient dropping to n H = 1.0 in the presence of 0.1 mM P i . The above results suggest a regulatory role of AMP and P i in the reconstitution of aspartate aminotransferase.