We found that Congo red agar allows identification of sporulation-deficientBacillus anthracis. Using Congo red agar, we isolated an asporogenic derivative of the protective antigen-producing strainB.anthracisdeltaSterne-1(pPA102). Polymerase chain reaction and Southern hybridization analyses of DNA from the asporogenic mutant revealed that a deletion was present inspoOA,an essential gene for the initiation of sporulation. The deletion also encompassed thespoIVBhomologue and a portion of therecNhomologue. The avirulentspoOAstrain deltaSterne-1(pPA102)CR4 is suitable for the safe production of protective antigen without endospore contamination of the vaccine production facility.Key words:Bacillus anthracis, protective antigen,spoOA, vaccine, Congo red.