Intramolecular isotope effects associated with the benzylic hydroxylation of a series of selectively deuterated isomeric xylenes and 4,4'-dimethylbiphenyl as catalyzed by various rat liver microsomal preparations and CYP2B1 were determined. Substrate analogs in which each methyl group contained either one (d2 substrates) or two (d4 substrates) deuterium atoms were used to determine the intrinsic isotope effect for the reaction. Specific values of the individual primary (P) and secondary isotope effects (S) were determined. P ranged from a low of 5.32 +/- 0.48 to a high of 7.57 +/- 0.42 depending upon the specific cytochrome P450 preparation used for catalysis. S had an average value of 1.03. The d3 substrates allowed exploration of the effect of distance on the magnitude of the observed isotope effect. The results indicate that the distance of 6.62 A that separates the carbon atoms of the para methyl groups of p-xylene is insufficient to suppress (mask) the intrinsic isotope effect for benzylic hydroxylation by all of the enzyme preparations examined. Conversely, a distance of 11.05 A, the minimal separation between the carbon atoms of the para methyl groups of p,p'-dimethylbiphenyl, is large enough to almost completely mask the intrinsic isotope effect for benzylic hydroxylation by the same set of enzymes.