Methotrexate (MTX) is a useful and often underutilized medication in the treatment of inflammatory bowel disease (IBD). However, there are no standard guidelines on MTX dosing in IBD patients. With azathioprine/6-mercaptopurine use, 6-thioguanine metabolites have been used to determine adequate dose and monitor compliance. MTX is an antifolate entering cells through the reduced folate carrier and activated to methotrexate polyglutamates (MTX PGs) by folylpolyglutamate synthase. The addition of these glutamic acid residues enhances intracellular retention of MTX and promotes the sustained inhibition of aminoimidazole carboxamide ribonucleotide (AICAR) transformylase and thymidylate synthase. These final steps in the de novo purine and pyrimidine biosynthesis results in the drugs antiproliferative and anti-inflammatory effects. Studies have demonstrated that individual pharmacogenetics and route of administration may play a significant role in MTX PGs and response to MTX in rheumatoid arthritis patients. MTX-PG levels of >60 nmol/L (compared to <60) were 5 fold more likely to have a good response and levels <20 nmol/L were 3 fold more likely to have a poor response compared in rheumatoid arthritis patients. The absorption of MTX is generally rapid, however variability in its absorption has been detected in subjects receiving oral treatment due to drug-induced epithelial denudation, motility changes and alterations in intestinal flora. Patients with IBD will often have mucosal changes, alterations in gut flora, and motility abnormalities that can affect drug absorption. No prior studies have evaluated the use of MTX-PG levels in IBD patients. To assess bioavailability of MTX in IBD patients from our center, we measured MTX-PG levels on those who received weekly MTX for greater than 3 months. Ten patients (9 Crohn’s disease and 1 Ulcerative Colitis) were evaluated. Eight patients were on SQ dosing (10 to 25 mg) and 3 were on PO dosing (25 mg). One patient had levels drawn on oral and after switching to SQ. The average MTX-PG was 25.8 nmoles/L in the PO group (n = 3) and 54.5 nmols/L in the SQ group (n = 8) (P = 0.002). Four patients (all in SQ group) had levels >60 nmoles/L and none had levels <20. The remainder of the patients were in the intermediate range (3 in PO, 4 in SQ) One patient whose oral MTX-PG levels were low (31.3) was switched from 25 mg PO to SQ which lead to higher MTX-PG levels(79.4). One patient with renal insufficiency intentionally on a lower SQ MTX dose of 10 mg/wk showed higher metabolite levels (69 nmols/L) due to lower renal excretion. Oral absorption of MTX may be poor in patients with inflammatory bowel disease. SQ MTX can lead to higher MTX metabolite levels, which may lead to improved therapeutic efficacy.