The sarcoplasmic reticulum-bound hexokinase of frog skeletal muscle has been solubilized and purified (PS) 60-fold. The molecular weight as obtained by calibrated Sephadex G-200 gel column and sucrose gradient analysis was 112 875 ± 5866 and 95 212 ± 2740 respectively. The propensity for molecular aggregation was considerable. The purified enzyme (PS) was rebound to enzyme-denuded sarcoplasmic reticulum. The kinetics of the original bound, purified unbound and purified rebound hexokinase was examined with respect to orthophosphate activation and inhibition with 1,5-anhydroglucitol-6-P (analogue of glucose-6-P ). At optimum orthophosphate concentration, activation was biphasic in the presence of varying MgATP for the original bound hexokinase. Thus orthophosphate activation at 0.5 and 10 mM MgATP was 90 and 100%, respectively. Solubilization and purification of the original bound hexokinase resulted in loss of the biphasic response with activation at high MgATP concentration only. Rebinding of the PS hexokinase to enzyme-denuded sarcoplasmic reticulum restored the biphasic activation in the presence of MgATP. The original sarcoplasmic reticulum-bound hexokinase was considerably more resistant to 1,5-anhydroglucitol-6-P inhibition with respect to MgATP, compared to the PS hexokinase. Rebinding of PS hexokinase to enzyme-denuded sarcoplasmic reticulum restored the resistance to 1,5-anhydroglucitol-6-P inhibition. Thus the K i for bound, unbound and rebound enzyme was 0.08, 0.026 and 0.11 mM respectively. Binding of hexokinase to sarcoplasmic reticulum (I/ K dissociation ) was optimal at pH 6.5 and 8.5. The dissociation constant, K D for original bound hexokinase at pH 6.5, 8.5 and 7.5 was 0.48, 0.54 and 63.4 units/ml, respectively.