Hydroxychloroquine (HCQ) is an antimalarial drug that is also used as a second-line treatment of rheumatoid arthritis (RA). Clinically, the use of HCQ is characterized by a long delay in the onset of action, and withdrawal of treatment is often a result of inefficacy rather than from toxicity. The slow onset of action can be attributed to the pharmacokinetics (PK) of HCQ, and wide interpatient variability is evident. Tentative relationships between concentration and effect have been made, but to date, no population PK model has been developed for HCQ. This study aimed to develop a population PK model including an estimation of the oral bioavailability of HCQ. In addition, the effects of the coadministration of methotrexate on the PK of HCQ were examined. Hydroxychloroquine blood concentration data were combined from previous pharmacokinetic studies in patients with rheumatoid arthritis. A total of 123 patients were studied, giving the data cohort from four previously published studies. Two groups of patients were included: 74 received hydroxychloroquine (HCQ) alone, and 49 received HCQ and methotrexate (MTX). All data analyses were carried out using the NONMEM program. A one-compartment PK model was supported, rather than a three-compartment model as previously published, probably because of the clustering of concentrations taken at the end of a dosing interval. The population estimate of bioavailability of 0.75 (0.07), n = 9, was consistent with literature values. The parameter values from the final model were: Cl = 9.9 +/- 0.4 L/h, V = 605 +/- 91 L, ka = 0.77 +/- 0.22 hours(-1), t(tag) = 0.44 +/- 0.02 hours. Clearance was not affected by the presence of MTX, and, hence, steady-state drug concentrations and maintenance dosage requirements were similar. A population PK model was successfully developed for HCQ.