To elucidate the mechanism by which hydroxychloroquine (HCQ) affects glucose metabolism, the effect of this drug and its enantiomers on insulin metabolism was studied using the cytosolic fraction of liver homogenates from healthy and diabetic rats. Eadie-Hofstee plots were monophasic suggesting that only a one-component enzyme system is involved in insulin degradation in the fraction used. Reaction velocity (V) vs substrate concentration plots were consistent with a Vmax model. HCQ caused a significant reduction in Vmax and Vmax/Km values in both healthy (Vmax, 3.63 +/- 0.46 vs 1.97 +/- 0.13, ng/min/mg; protein P < 0.001; and Vmax/Km 0.265 +/- 0.015 vs 0.112 +/- 0.004, ml/min/g protein) and diabetic rats (Vmax, 0.718 +/- 0.06 vs 0.360 +/- 0.024, ng/min/mg protein; and Vmax/Km, 0.05 +/- 0.002 vs 0.023 +/- 0.001, ml/min/g protein). Significant reduction in the V was observed in the presence of racemic (rac)-, R-, or S-HCQ. Ranking of the inhibitory potency was HCQ > S = R except at highest examined concentration (20 mg/mL) which was HCQ > S > R. In conclusion, the effect of HCQ on insulin degradation appears to be, in part, through inhibition of cytosolic insulin metabolizing enzyme. The effect is not stereoselective except at high concentrations. The R- and S-HCQ may have synergistic effects on inhibition of insulin degradation.