The rate of hydrolysis of N-(phenylacetyl)glycyl-D-valine (PAGV), an acyclic penicillin G analogue, at pH 0, 1, 3, 5, 7, 9, 11, 13, and 14 has been measured at 37 °C and a pH-rate profile constructed. At each pH, hydrolysis of both the (phenylacetyl)glycyl amide bond and glycyl-D-valine peptide bond was monitored. At pH 3, 5, 7, 9, and 11, the hydrolysis products glycyl-D-valine and D-valine were derivatized with naphthalene-2,3-dialdehyde in the presence of cyanide; the resultant 1-cyano-2-substituted-benz[f]isoindole (CBI) derivatives, which are highly fluorescent, were then quantified using reverse-phase HPLC. The hydrolysis reactions were explicitly shown to be first-order in peptide concentration at pH 5 and 9, and all rates were shown to be independent of the buffer concentration. The rates at pH 0, 1, 13, and 14 were measured in 1 M DCl, 0.1 M DCl, 0.1 M NaOD, and 1 M NaOD, respectively, and the hydrolysis products were detected by 1 H NMR. The first-order rate constants obtained from the above reactions were fit to the general equation kto yield the following results: for hydrolysis of the (phenylacetyl)glycyl bond, k H2O ) (9.05 ( 6.36) × 10 -11 s -1 , k H3O + ) (1.60 ( 1.04) × 10 -6 M -1 s -1 , and k OH -) (1.11 ( 0.73) × 10 -6 M -1 s -1 ; and for hydrolysis of the glycyl-D-valine bond, k H2O ) (8.23 ( 4.33) × 10 -11 s -1 , k H3O + ) (1.67 ( 0.80) × 10 -6 M -1 s -1 , and k OH -) (1.16 ( 0.56) × 10 -6 M -1 s -1 . At pH 7, the hydrolysis of both the (phenylacetyl)glycyl amide bond and glycyl-D-valine peptide bond is dominated by k H2O . The corresponding half-life for (phenylacetyl)glycyl bond hydrolysis is 243 years (with a range of 143-817 years within experimental error), while that for glycyl-D-valine bond hydrolysis is 267 years (with a range of 175-564 years).