We devised an effective purification for the microdetermination of prostaglandin E2 (PGE2) in human joint fluid using gas chromatography/selected ion monitoring and determined PGE2 in the joint fluid in rheumatoid arthritis (RA) patients using this method. The methyl estermethoxime-tert-butyldimethylsilyl ether derivative was prepared, then gas chromatography/selected ion monitoring was carried out by monitoring the ion at m/z 566.4 for PGE2 and at m/z 570.4 for the internal standard (PGE2-d4). A good linear response over the range of 10 pg to 50 ng was demonstrated. We detected PGE2 to a level of about 46 pg/ml in the joint fluid of RA patients. The level of PGE2 in RA patients was significantly higher than that in osteoarthritis patients used as controls. Moreover, we measured inflammatory cytokine (IL-1beta, TNFalpha, IL-6 receptor) levels in joint fluid by using enzyme-linked immunosorbent assay. A relationships between the PGE2 level in joint fluid and these cytokines or biochemical data as the indicator of RA disease was not observed. We found that the PGE2 level in each patient was influenced by therapeutic drugs. The PGE2 level in RA patients with non-steroidal anti-inflammatory drugs was lower than with steroids.