Experiments were designed to study chlorine dioxide (CD) gas sterilization under square-wave conditions. By using controlled humidity, gas concentration, and temperature at atmospheric pressure, standard biological indicators (BIs) and spore disks of environmental isolates were exposed to CD gas. The sporicidal activity of CD gas was found to be concentration dependent. Prehumidification enhanced the CD activity. The D values (time required for 90% inactivation) of Bacillus subtilis subsp. niger ATCC 9372 BIs were estimated to be 1.5, 2.5, and 4.2 min when exposed to CD concentrations of 30, 15, and 7 mg/liter, respectively, at 23 degrees C and ambient (20 to 40%) relative humidity (RH). Survivor tailings were observed. Prehumidification of BIs to 70 to 75% RH in an environmental chamber for 30 min resulted in a D value of 1.6 min after exposure to a concentration of 6 to 7 mg of CD per liter at 23 degrees C and eliminated survivor tailing. Prolonging prehumidification at 70 to 75% RH for up to 16 h did not further improve the inactivation rate. Prehumidification by ultrasonic nebulization was found to be more effective than prehumidification in the environmental chamber, improving the D value to 0.55 min at a CD concentration of 6 to 7 mg/liter. Based on the current observations, CD gas is estimated, on a molar concentration basis, to be 1,075 times more potent than ethylene oxide as a sterilant at 30 degrees C. A comparative study showed B. subtilis var. niger BIs were more resistant than other types of BIs and most of the tested bacterial spores of environmental isolates.