We evaluated the safety and immunogenicity of attenuated Shigella flexneri 2a vaccine candidate CVD 1203, which harbors precise deletions in the plasmid gene virG and in the chromosomal gene aroA. CVD 1203 invades epithelial cells but undergoes minimal intracellular proliferation and cell-to-cell spread. Fasting healthy volunteers, aged 18 to 40 years, were randomly allocated (double-blind design) to receive either CVD 1203 vaccine or placebo, along with sodium bicarbonate buffer, on days 0 and 14, as follows. At the time of the first inoculation, 10 subjects received placebo (group 1) and 22 subjects received either 1.5 x 10(8) (group 2; 11 subjects) or 1.5 x 10(9) (group 3; 11 subjects) CFU of CVD 1203. Fourteen days later, subjects from group 1 received 1.2 x 10(6) CFU of CVD 1203 and subjects from groups 2 and 3 received 1.2 x 10(8) vaccine organisms. Clinical tolerance was dose dependent. After a single dose of CVD 1203 at 10(6), 10(8), or 10(9) CFU, self-limited (<48-h duration) objective reactogenicity (fever, diarrhea, or dysentery) developed in 0, 18, and 72% of subjects, respectively, and in no placebo recipients. CVD 1203 induced immunoglobulin G seroconversion to S. flexneri 2a lipopolysaccharide (LPS) in 30, 45, and 36% of subjects from groups 1, 2, and 3, respectively, and stimulated immunoglobulin A-producing anti-LPS antibody-secreting cells in 60, 91, and 100% of subjects, respectively. After vaccination, significant rises in tumor necrosis factor alpha concentration in serum (groups 1, 2, and 3) and stool (group 2) samples were observed. We conclude that engineered deletions in virG and aroA markedly attenuate wild-type S. flexneri but preserve immunogenicity; however, less reactogenic vaccines are needed.