Degradation of penicillin in acidic media (pH 2.7) was monitored by high-pressure liquid chromatography and UV spectroscopy. The effects of temperature, buffer concentration, and ionic strength were examined. A degradation pathway is proposed, and the apparent first-order rate constant and energy of activation were calculated for each reaction. One or more degradation products containing a sulfhydryl group, a functional group often suggested as having a major role in eliciting allergic responses to penicillin therapy, were present throughout the degradation scheme.