Salmonella enterica serovar Typhimurium utilizes molecular hydrogen as a substrate in various respiratory pathways, via H 2 -uptake enzymes termed Hya, Hyb, and Hyd. A different hydrogenase, the hydrogen-evolving Hyc enzyme, removes excess reductant during fermentative growth. Virulence phenotypes conferred by mutations in hyc genes, either alone or in combination with mutations in the H 2 -uptake enzyme genes, are addressed. Anaerobically grown ⌬hycB or ⌬hycC single-deletion strains were more sensitive to acid than the wild-type strain, but the ⌬hyc strains were like the virulent parent strain with respect to both mouse morbidity and mortality and in organ burden numbers. Even fecal-recovery numbers for both mutant strains at several time points prior to the animals succumbing to salmonellosis were like those seen with the parent. Neither hydrogen uptake nor evolution of the gas was detected in a hydrogenase quadruple-mutant strain containing deletions in the hya, hyb, hyd, and hyc genes. As previously described, a strain lacking all H 2 -uptake ability was severely attenuated in its virulence characteristics, and the quadruple-mutant strain had the same (greatly attenuated) phenotype. While H 2 levels were greatly reduced in ceca of mice treated with antibiotics, both the ⌬hycB and ⌬hycC strains were still like the parent in their ability to cause typhoid salmonellosis. It seems that the level of H 2 produced by the pathogen (through formate hydrogen lyase [FHL] and Hyc) is insignificant in terms of providing respiratory reductant to facilitate either organ colonization or contributions to gut growth leading to pathogenesis.