The sporicidal activity of chlorhexidine gluconate in aqueous and alcoholic solution against spores of Bacillus subtilis was examined over a broad temperature range. Activity was not observed at 20 degrees C even with concentrations as high as 10% chlorhexidine. Temperatures of 37 degrees-70 degrees C in combination with such high concentrations were required for reductions in spore viability. No viable spores were recoverable after 4 h contact at 55 degrees C with 10% aqueous chlorhexidine and none after 3 h contact with the alcoholic solution. Because of the high concentrations necessary for activity and the possibility of sporostasis occurring from inefficient chlorhexidine inactivation, existing inactivation systems were examined and modified to obtain satisfactory results. The spores of other Bacillus species examined (B. cereus, B. megaterium and B. stearothermophilus) proved to be considerably less resistant than those of B. subtilis. Presence of organic matter had little effect on the activity.