Dipyrone is a non-steroidal anti-inflammatory drug. It is a widely used and well tolerated analgesic drug which is however compromised by agranulocytosis as adverse effect.. The complex metabolism of dipyrone has been subject of many invivo studies. However, the specific cytochrome P450 enzymes involved catalysing the formation of 4- aminoantipyrine (4-AA) from 4-methylaminoantipyrine (4-MAA) is still not unequivocally identified. The aim of the present study was to identify the cytochrome P450 enzyme (CYP) mediating this reaction. The relevant CYP was identified using virus expressed isolated rat liver microsomes with chemical inhibition studies. The substrate of 4- methylaminantipyrine was employed at six different concentrations (25, 50, 100, 400, 800 and 1200 μmol/l) with 100 μmol/l of selective inhibitors of CYP1A2 (furafylline, fluvoxamine), CYP3A4 (ketoconazole), CYP2A6 (coumarin), CYP2D6 (Quinidine), CYP2C19 (omeprazole), CYP2C9 (sulphaphenazole) and CYP1A1 (alpha - naphthoflavone). 4-MAA and 4-AA were then analyzed by HPLC. The formation rates corresponding to the six substrate concentrations were subjected to a regression analysis in order to estimate KM and Vmax according to the Michaelis-Menten equation by nonlinear regression analysis with the program Sigma Plot. The results clearly demonstrated that the N-demethylation of 4-MAA by microsomes prepared from baculovirus-expressing CYP was pronounced with CYP2C19. Intrinsic clearance of the most active enzymes were 0.092, 0.027, and 0.026 for the CYP enzymes 2C19, 2D6 and 1A2 respectively. Metabolism by rat liver microsomes was strongly inhibited by omeprazole (IC50 of 0.05). The outcome of present study concluded that the enzyme CYP2C19 apparently has an important role in N-demethylation of 4- methylaminoantipyrine.