The objective of the current study was to investigate the metabolism of cytochrome P450 (CYP) probe substrates in male Sprague Dawley rat liver microsomes and to determine their substrate specificities. Time and microsomal protein concentrations were varied to determine the linear conditions for each reaction. Appropriate substrate concentrations were chosen to determine the apparent K(m) and V(max) for 17 different reactions under initial rate conditions of protein and reaction time. All reactions appeared to follow Michaelis-Menten kinetics. Subsequently, each substrate was incubated at one to two times K(m) with each of 14 baculovirus cDNA-expressed rat CYP enzymes to determine the specificity of the reaction monitored. Of the 14 enzymes tested, seven were seen as the major rat CYP enzymes responsible for the majority of the substrate metabolism tested. Testosterone 2alpha- and 16alpha-hydroxylation reactions were conducted primarily by CYP2C11, and midazolam 4-hydroxylation and triazolam 1'-hydroxylation were preferentially catalyzed by CYP3A1/2, but specificity was otherwise generally poor. The results presented herein clearly indicate that care must be taken in interpretation of metabolism results obtained in rats using standard probe substrates, especially in extrapolation of those results to humans.