Specific confirmatory tests are needed to identify and quantify beta-lactam antibiotic residues detected in milk at levels of less than 10 parts per 10(9) (ppb) by screening tests. A liquid chromatographic method for penicillin G was developed using the liquid chromatography system for cleanup as well as analysis. Milk was deproteinized with two volumes of acetonitrile. The acetonitrile was extracted with hexane-methylene chloride (1:1) and the remaining water layer was concentrated by evaporation. The water layer (2 ml = 5 ml milk) was injected onto a Polymer Laboratories PLRP-S column using a WISP autosampler with the solvent, 0.01 M pH 7.0 phosphate buffer (A). Penicillin G was eluted with acetonitrile (B) gradient A-B (100:0) (0-3 min)-(40:60) (25 min). Penicillin G eluted as a narrow band in less than 0.5 min. A narrow fraction containing penicillin G was collected and rechromatographed on the same type of column at low pH (1.96). This effectively separated penicillin G from interferences. Recoveries were 92 +/- 9% with a sensitivity limit near 2 ppb. The approach used is applicable to determination of other beta-lactam antibiotics but specific conditions for analysis must be determined for each one. The cleanup procedure can be automated using an autosampler, gradient controller, and fraction collector.