A series of experiments was conducted to determine growth and toxin formation by proteolytic strains of Clostridium botulinum in broth media that have known pH values (5-7), NaCl concentrations (0-4%), and controlled oxygen-nitrogen atmospheres. Lower pH and higher NaCl levels inhibited growth and toxin production by vegetative cells, but 15% oxygen in the headspace was insufficient for inhibition in all media. When spores were used as inocula and the tubes were gas flushed, outgrowth and toxin production generally occurred only under a 1% or less oxygen atmosphere. Occurrence of growth and toxin was favored by high pH and low NaCl levels and was related to spore inoculum size. Spores were also inoculated into a mixed fermenter with controlled oxygen levels in the headspace. Times to measurable turbidity increased with greater oxygen levels from 36 h at 0.005% O2 to 109 h at 0.7% O2; however, growth rates were unaffected by headspace oxygen levels. No toxin was observed with 0.9% O2, further demonstrating that the critical level of oxygen for germination and growth is approximately 1%.