Thymol stimulated O2- production in guinea pig neutrophils. O2- production occurred about 30 sec after the addition of thymol, and its rate was independent of extracellular Ca2+. Thymol-induced activity was inhibited by trifluoperazine (TFP), an inhibitor of protein kinase c, and its IC50 was less than that for 12-O-tetradecanoyl phorbol 13-acetate (TPA) induced activity. After complete activation, O2- production was reversed by addition of TFP or by washing out and resuspending in a stimuli-free medium. The responsiveness of the thymol-pulsed cells to another stimulus, TPA, was somewhat more than resting cells, but the responsiveness of the former cells to thymol was about half that of the latter cells. The ATP level of cells was reduced to one half its initial value during activation by thymol. These data suggest that the magnitude of thymol-induced O2- production in neutrophils is dependent on the initial density of the binding sites of the cells with thymol and the initial intracellular ATP concentration.