Human whole saliva contains the hypothiocyanite ion (OSCN-) which is the principal antimicrobial product of the salivary peroxidase system. The peroxidase system requires a source of peroxide in order to produce OSCN- and in the human mouth this source has been assumed to be primarily the peroxidogenic oral bacteria. However, we report here studies which show that samples of stimulated human parotid saliva collected directly from Stenson's duct have concentrations of OSCN- which are similar to those found in human whole saliva. Thus, the peroxidogenic bacteria are not an absolute requirement for the generation of significant levels of OSCN- in the human mouth. Supplementation of human whole saliva with components [thiocyanite (SCN-), hydrogen peroxide (H2O2)] of the peroxidase system produces a 10-fold or greater increase in OSCN- concentration. However, the magnitude of this increase is critically dependent upon pH and upon the relative and absolute concentrations of SCN- and H2O2. The pH dependence of OSCN- generation is similar for human whole saliva and for the lactoperoxidase/SCN-/H2O2 system. The optimum is in the range 6.5-7.0. Samples of parotid saliva adjusted to pH 6.5 and supplemented with appropriate amounts of SCN- and H2O2 show increases in OSCN- concentrations which are similar to those observed with whole saliva. The results show that there is a significant source of H2O2 within the parotid gland, that the OSCN- generating potential of parotid saliva is similar to that of whole saliva and that the enhancement of OSCN- levels in saliva by addition of SCN- and H2O2 is critically dependent upon pH and upon the relative and absolute concentrations of H2O2 and SCN-.