Biochemical tests commonly used to screen for Vibrio cholerae in environmental samples were evaluated, and we found that a combination of alkaline peptone enrichment followed by streaking on thiosulfate citrate bile salts sucrose agar and testing for arginine dihydrolase activity and esculin hydrolysis was an effective rapid technique to screen for aquatic environmental V. cholerae. This technique provided 100% sensitivity and > or =70% specificity.