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Introduction Quantification of cytochrome P 450 is a major issue in the development of new drugs. Different assays have been reported, but few are very selective for the 3A isoform or cytochrome P 450. The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor 3-[(3, 4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-one has recently been used successfully to probe isoform 3A of cytochrome P 450 in the liver. However, its selectivity for the rat isoform remains to be established as well as its applicability in other tissue, such as the intestine. The purpose of this study was to ascertain the specificity of this substrate for the rat 3A isoform of cytochrome P 450 using Supersomes and its application in non-hepatic tissue (e.g., intestine). Methods: Specificity of the 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one for the isoform 3A of rat cytochrome P 450 was established by using either isoform-specific inhibitory antibody or microsomes expressing only one cytochrome P 450 isoform. Activity was assayed in rat liver and intestinal microsomal protein preparations. Results: Experiments with inhibitory antibodies revealed that in liver and intestinal microsomes, more than 90% of the substrate metabolism was inhibited by antibodies against isoform 3A. Selectivity of the substrate for rat 3A isoform was further determined by testing the metabolic activity of various Supersomes™ preparations. Discussion: In conclusion, our results validate the usefulness of 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one as a simple and specific substrate to study the activity of the isoform 3A of cytochrome P 450 in the rat liver and intestine.