Concentrations of vancomycin in serum were measured by an automatic high-performance liquid chromatographic (HPLC) micromethod. Vancomycin is a glycopeptide antibiotic with broad application in the therapy of gram-positive infections. As this drug is potentially nephro- and ototoxic, a method to maximize its therapeutic benefit while minimizing the risk of toxicity is desirable. This fully automated HPLC method did not involve a sample pretreatment step. The configuration of the apparatus permitted a solid phase extraction of the serum sample on two precolumns filled with a reversed-phase material, followed by a chromatographic separation of the sample constituents on an analytical column. The reversed phase analytical column (muBondapak C18) was flushed with a mobile phase of water-acetonitrile-triethylamine, 870: 130: 4 (vol/vol/vol); the pH was adjusted to 3.0 with orthophosphoric acid. Precision was expressed as the coefficient of variation (CV), which was always < or = 4.13% for intra- and inter-assays (n = 10) in the range 2-50 micrograms/ml. We compared this specific HPLC determination to an enzyme-multiplied immunoassay (EMIT). Fifty clinical samples obtained from patients under vancomycin therapy were assayed by each method and results compared using a linear regression analysis. There was a significant correlation between results from HPLC and EMIT: EMIT = 0.51 + 1 x HPLC (r = 0.963; p < 0.0001). The rapidity and specificity of this HPLC micromethod make it suitable for use in the monitoring of serum levels of vancomycin and for use in pharmacokinetic studies of this antibiotic.