We used 72 environmental non– B. anthracis bacilli to validate methods for ruling out B. anthracis. Most effective were horse blood agar, motility testing after a 2-h incubation in trypticase soy broth, and screening with a B. anthracis –selective agar. Keywords: Bacillus anthracis, PCR, motility, hemolysis, bioterrorism, PLET agar.