Objective. MTX is a prodrug producing anti-arthritic effects through a folylpolyglutamate synthase- mediated activation to MTX polyglutamates (MTXPGs). Our objective was to characterize the pharmaco- kinetics of intracellular MTXPGs and the factors associated with their accumulation in adult RA patients treated with MTX weekly. Methods. MTX pharmacokinetics were evaluated in 47 MTX-naive patients enrolled in an MTX dose- escalation study for an average of 20 weeks and 223 patients enrolled in a cross-sectional study under long-term MTX therapy. Short-chain (MTXPG12), long-chain (MTXPG3) and very long-chain (MTXPG45) concentrations were measured in circulating red blood cells using liquid chromatography. Statistical ana- lyses consisted of non-linear mixed models, multivariate regression analyses and Wilcoxon signed-rank test. Results. The accumulation of MTXPG15 was sigmoidal and steady-state concentrations were achieved after 7 weeks of therapy. However, additional exposure and MTX dosage escalation produced a selective redistribution towards longer chain MTXPGs at the expense of shorter chain MTXPGs. Age, glomerular filtration rate and route of MTX administration were the most important predictors of MTXPG accumula- tion. In 10 patients, a switch from oral to parenteral MTX was associated with a 37% increase in long- chain MTXPGs, a 132% increase in very long-chain MTXPGs and a concomitant 31% reduction in disease activity (P < 0.02). Conclusion. The selective emergence of long-chain MTXPGs is function of dose, time of exposure and hence dosage intensity. Switching from oral to parenteral MTX produces a selective accumulation of longer chain MTXPGs that are known to be more potent inhibitors of de novo purine biosynthesis than shorter chain MTXPGs.