BACKGROUND: Methotrexate is an immunomodulatory drug for patients with Crohn's disease. Erythrocyte MTX-polyglutamates (MTX-PG(1-5)) may be used for therapeutic drug monitoring (TDM) as MTX-PG is thought to mediate MTX's efficacy. Information on determinants of the concentration of MTX-PG in patients with Crohn's disease is lacking. We aim to identify clinical and biochemical determinants of the erythrocyte MTX-PG(1-5) and MTX-PG(total) concentration in patients with Crohn's disease. METHODS: Adults with Crohn's disease on methotrexate treatment who visited the outpatient clinic of Amsterdam UMC were included. Erythrocyte MTX-PGs were measured by tandem mass spectrometry. RESULTS: Nineteen patients were included, with a median duration of MTX use of 77 months (range 7-202). Twelve patients received MTX monotherapy, whereas 7 patients were on concomitant TNF-alpha inhibitors. The mean dose of MTX was 15.5 mg (SD +/- 2.8) and 12 (63%) patients used subcutaneous MTX. MTX-PG(1-5) were successfully measured in 18 patients, showing substantial variability in concentrations of MTX-PG(total) and individual species. The median MTX-PG(total) was 117.1 nmol/L (range 46.4-258.7) with preferential accumulation of MTX-PG(3) (43.1 nmol/L, range 15.3-96.1). Patients on subcutaneous compared to oral MTX had higher median MTX-PG((4,5)) levels (55 versus 9 nmol/L, p = 0.01). Higher age (beta = 0.71) and lower estimated glomerular filtration rate (beta = - 0.52) were associated with a significantly higher MTX-PG(total) concentration (R(2) = 0.60, p = 0.001). CONCLUSION: MTX-PG concentrations display a considerable inter-individual variability. Higher MTX-PG accumulation is associated with subcutaneous administration, higher age, and lower renal function in Crohn's disease patients.