Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.