Reverse phase high-performance liquid chromatography with ultra-violet detection has been used for the simultaneous identification and determination of ampicillin, cloxacillin and some of their degradation products following their extraction from an intramammary preparation used in veterinary medicine. Extraction of the intramammary base into petroleum ether (40–60°C) and partition of the penicillins into the mobile phase gave a mean recovery of 100.8% for ampicillin with coefficient of variation of 1.0 and 103.4% for cloxacillin with coefficient of variation of 1.2 (n=6). Using optimised HPLC conditions ampicillin eluted in approximately 2 min and cloxacillin in approximately 8 min. The overall method was found to be stability indicating, since cloxacillin and ampicillin eluted independently of their degradation products.