The saprophytic Paenibacillus and Bacillus spp. found in cooked chilled foods may have an effect on the growth of Clostridium botulinum, a major microbiological hazard, especially for pasteurized vacuum-packaged products. Culture supernatants of 200 strains of Paenibacillus and Bacillus strains isolated from commercial cooked chilled foods containing vegetables were screened for activity against C. botulinum type A, proteolytic type B, and type E strains in a well diffusion assay. Nineteen strains were positive against C. botulinum. Among those, seven Paenibacillus polymyxa strains showed the highest antibotulinal activity and the largest antimicrobial spectrum against C. botulinum strains. The antibotulinal activity was evaluated throughout the growth of a representative strain of the positive P. polymyxa strains. The antimicrobial activity was detected in the culture supernatant from late-log/early stationary phase of the bacteria, which occurred after 7 to 10 days of incubation at 10 degrees C and after 2 to 3 days at 20 degrees C in nutrient broth and in vegetable purées under aerobic or anaerobic conditions. In co-cultures with the positive strain of P. polymyxa in nutrient broth and vegetable purées, a C. botulinum type E strain was inhibited whenever P. polymyxa reached stationary phase and produced its antimicrobial activity before C. botulinum began its exponential growth phase. The antimicrobial activity of P. polymyxa against C. botulinum was attributed to the production of antimicrobial peptides resistant to high temperature and acidity. Other gram-positive and -negative bacteria (Escherichia coli, Streptococcus mutans, Leuconostoc mesenteroides, and Bacillus subtilis) were also sensitive to these antimicrobial peptides.