Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight (M-w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes (M-w/M-n) of 1.3-1.7. Kinetic plots of 1/M-w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant (k(h)) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (In k(h) vs 1/T), with an activation energy (E-a) of 137 kJ/mol and Arrhenius constant (A) of 7.86 x 10(15) h(-1). H-1 NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de-N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1 -> 4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < pK(a,GlcA). The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the pK(a) of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.