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The lifetimes of fluorescent components of matrix NADH in isolated porcine heart mitochondria were investigated using time-resolved fluorescence spectroscopy. Three distinct lifetimes of fluorescence were resolved: 0.4 (63%), 1.8 (30%), and 5.7 (7%) ns (% total NADH). The 0.4 ns lifetime and the emission wavelength of the short component were consistent with free NADH. In addition to their longer lifetimes, the remaining pools also had a blue-shifted emission spectrum consistent with immobilized NADH. On the basis of emission frequency and lifetime data, the immobilized pools contributed >80% of NADH fluorescence. The steady-state kinetics of NADH entering the immobilized pools was measured in intact mitochondria and in isolated mitochondrial membranes. The apparent binding constants (K(D)s) for NADH in intact mitochondria, 2.8 mM (1.9 ns pool) and >3 mM (5.7 ns pool), were on the order of the estimated matrix [NADH] (approximately 3.5 mM). The affinities and fluorescence lifetimes resulted in an essentially linear relationship between matrix [NADH] and NADH fluorescence intensity. Mitochondrial membranes had shorter emission lifetimes in the immobilized poo1s [1 ns (34%) and 4.1 ns (8%)] with much higher apparent K(D)s of 100 microM and 20 microM, respectively. The source of the stronger NADH binding affinity in membranes is unknown but could be related to high order structure or other cofactors that are diluted out in the membrane preparation. In both preparations, the rate of NADH oxidation was proportional to the amount of NADH in the long lifetime pools, suggesting that a significant fraction of the bound NADH might be associated with oxidative phosphorylation, potentially in complex 1.