A convenient and sensitive high-performance liquid chromatographic assay for the simultaneous quantitation of lidocaine and its four metabolites has been developed. The samples containing lidocaine and its metabolites were eluted from a microparticulate octadecylsilane column using a mobile phase of 0.1 M phosphate buffer (pH 3.0) containing 10% acetonitrile. This method was applied to studies on in vitro metabolism and in vivo pharmacokinetics of lidocaine in rats. Kinetic studies of in vitro microsomal metabolism of lidocaine indicated that the apparent Km and Vmax for aromatic hydroxylation were smaller than those for N-deethylation. Lineweaver—Burk plots of the N-deethylation of lidocaine and those of its two primary metabolites indicated that at least two isozymes are taking part in these reactions. In in vivo lidocaine pharmacokinetics, the area under the blood concentration—time curve for the monodeethylated product, ω-ethylamino-2,6-dimethylacetanilide (1), varied considerably depending on the route of administration.