The aqueous degradation of daptomycin, a lipopeptide antibiotic, was investigated as a function of substrate concentration (0.5-10.0 mM), pH (9.0-10.5), buffer concentration (0.06-0.20 M borate, glycinate, or carbonate buffers), temperature (20-50 degrees C), and ionic strength (0.1-0.8). The primary degradation pathway was determined by electrospray-mass spectroscopy (ES-MS), Fourier transform infrared (FTIR), and fluorescence spectroscopy to be hydrolysis of the ester linkage between the C-terminus (kynurenine) and the side chain of the fourth residue (threonine). The reaction was first order with respect to time; however, the reaction order with respect to substrate concentration was 1 mM. Insignificant buffer effect was observed. The reaction was subject to specific base catalysis. Activation parameters were E(a) = 13.6 kcal/K.mol, DeltaH++ = 13.0 kcal/K.mol, and DeltaS++ = -19.2 eu. The positive primary salt effect was observed with negative deviation at high concentration of salt. The magnitude of the salt effect depended on salt identities in the order sodium < potassium < calcium chloride.