The anthrax toxins and capsule, encoded by plasmids pX01 and pX02, respectively, are the only known virulence factors of Bacillus anthracis and are considered essential for full virulence. Some B. anthracis strains cured of pX01, such as delta Ames-1, remained virulent for mice. The virulence was partially mediated by pX02, as determined by phage transduction. pX02 plasmids from the delta Ames-1 and Pasteur strains were mutagenized with transposon Tn917 to identify loci associated with virulence. The capsule phenotype, virulence and pX02 restriction pattern of the insertion mutants were characterized. Two mutants that produced no detectable capsule were avirulent. One had a deletion of more than 20 kb, which included the structural genes required for capsule synthesis (cap); the second had an insertion outside of cap. Two mutants with reduced encapsulation had insertions at different sites outside cap and were less virulent, whereas one that was normally encapsulated, but had a high rate of pX02 curing, was unaltered in virulence. Mutants that produced greater amounts of capsule than the parental strain were more virulent, and a few that produced wild-type levels of capsule were less virulent.