Various skin test reagents supplying minor determinants for detecting penicillin hypersensitivity have been examined by high-performance liquid chromatography (HPLC) for composition and stability. HPLC systems capable of separating and determining the four diastereoisomers of benzyl-D-penicilloic acid and the two benzyl-D-penicilloic acids were developed for this purpose. The "simple skin test reagent," consisting of an aged partial alkaline hydrolysate of penicillin, is possibly an adequate source of (5R,6R)-benzyl-D-penicilloate whereas the "simple skin test reagent," consisting of aged aqueous solution of penicillin, is a questionable source of this compound. A modified Levine, Voss, Redmond, and Zolov minor determinant mixture (MDM) reagent and the components (5R,6R)-benzyl-D-penicilloate and (5R)-benzyl-D-penilloate have been found to be highly labile in aqueous solution, giving rise to a mixture of diastereoisomers. The tendency to epimerize at C-5 was a prominent feature of (5R,6S)- and (5S,6R)- as well as (5R,6R)-benzyl-D-penicilloic acids. The MDM reagent has been prepared in single-dose ampules as a dried, lyophilized powder that can be stored without change and used as needed. Lyophilized MDM has served as a satisfactory substitute for freshly prepared MDM in several individuals with MDM-positive history and, in a recent clinical study, evaluating the question of penicillin skin test sensitization. This convenient, stable, single-dose form of the MDM reagent should facilitate skin testing for penicillin sensitivity.