Treatment of high-molecular-weight hyaluronan (HA) with peroxynitrite at neutral pH (ONOO-/ONOOH) results in altered mobility on agarose gel electrophoresis, as well as reduced limiting viscosity number. Both effects are consistent with a reduction in HA molecular weight. HA is protected from peroxynitrite attack to varying extents by addition of alternate target molecules. Thiourea is extremely effective as a protective agent, dimethyl sulfoxide is moderately effective, while sodium benzoate and mannitol are slightly effective. A similar pattern of protection is observed when HA is degraded by hydroxyl radical generated by a metal ion/hydrogen peroxide system. On the basis of these observations, peroxynitrite is proposed to have hydroxyl radical-like activity in degrading HA.