Hydroxychloroquine (HCQ) is a racemic antiarthritic agent that has a long half-life (t1/2) in plasma and accumulates in blood cells. To study the relationships between HCQ concentrations in plasma, serum, and whole blood and to determine the optimal blood fraction to use for therapeutic drug monitoring of the drug, we studied the relative distribution of the HCQ enantiomers in various fractions of human blood under in vivo and in vitro conditions. Substantially greater concentrations of both enantiomers were found in serum as compared with plasma because of release via platelet activation. After in vitro incubations of the separated blood cells with HCQ, high concentrations of both enantiomers were found in leukocytes, and low concentrations in erythrocytes and platelets; the R:S ratio in vitro was near unity in all of the cells examined. Unlike the in vitro cellular uptake, the concentrations of HCQ in vivo were significantly lower and stereoselective (R:S ratio = 2). There was almost no drug in the polymorphonuclear cells (PMN) in vivo, despite a substantial uptake in vitro after incubation of separated cells. The enantiomeric (R:S) ratio in the urinary excretion of the enantiomers was significantly correlated with that in plasma. The plasma-protein binding of the enantiomers was stereoselective and complimented the cellular uptake findings; the unbound fraction was dependent on the plasma concentrations of alpha 1-acid glycoprotein, but not albumin. Although concentrations in whole blood correlated well with those in lymphocytes and monocytes (the proposed site of HCQ action), stronger correlations were found between concentrations in serum and in the mononuclear cells.