To determine the safety of fresh-cut vegetables packaged in modified atmosphere, challenge studies using both nonproteolytic and proteolytic strains of Clostridium botulinum were performed with a variety of fresh-cut packaged salads and vegetables stored at different temperatures. When vegetables were inoculated with spores of C. botulinum and incubated in low-O2 atmospheres, spore germination and growth and toxin production were observed. Botulinum toxin was produced by proteolytic types A and B on onion, butternut squash, rutabaga, salad, and stir-fry vegetables. Nonproteolytic C. botulinum produced toxin on butternut squash and salad. Nonproteolytic C. botulinum was capable of producing neurotoxin at temperatures as low as 5 degree C whereas proteolytic strains produced neurotoxin at 15 degrees C and higher. Although most samples were visibly spoiled before detection of botulinum toxin, samples of butternut squash and onion remained acceptable after detection of toxin. The strict maintenance of low temperatures (< 5 degrees C) is recommended in order to control the potential growth of C. botulinum on fresh-cut vegetables packaged in a modified atmosphere.