NMR studies of the binding of a substrate to an inactive HIV-1 protease construct, containing an active site mutation PR(D25N), are reported. Substrate titration measurements monitored by HSQC spectra and a (15)N-edited NOESY experiment show that the chromogenic substrate analog of the capsid/p2 cleavage site binds to PR(D25N) with an equilibrium dissociation constant, K(D), of 0.27 +/- 0.05 mM, and upper limits of the association and dissociation rate constants, 2 x 10(4) M(-1)s(-1) and 10 s(-1), respectively, at 20 degrees C, pH 5.8. This association rate constant is not in the diffusion limit, suggesting that association is controlled by a rare event, such as opening of the protease flaps. Analysis of (15)N relaxation experiments reveals a slight reduction of S(2) values in the flap region, indicating a small increase in the amplitude of internal motion on the sub-nsec timescale. In addition, several residues in the flap region are mobile on the conformational exchange timescale, msec-microsec. Flap dynamics of the protease-substrate complex are compared with those of protease-inhibitor complexes, and the implications of these results for substrate-binding models are discussed.