High-performance liquid chromatographic methods for determination of the isomers of moxalactam in plasma and urine have been developed. Conventional reverse-phase chromatography was used for plasma assays, and an ion-pairing reagent was included for urine assays. Detection limits were 1.5 micrograms/ml of plasma and 7.5 micrograms/ml of urine. The high-performance liquid chromatographic assays were extensively compared with a microbiological assay (detection limit, 1 microgram/ml), using samples from human volunteers to whom moxalactam had been administered as well as plasma and urine from untreated humans, to which moxalactam was added. The correlations between the assays were quite good, but the precision and accuracy of the high-performance liquid chromatographic methods were superior. Both types of assays were used in a study of the stability of moxalactam-containing samples at various temperatures.