ABSTRACT Salmonella enterica serovar Typhimurium possesses a multi-copper-ion oxidase (multicopper oxidase), CueO (also known as CuiD), a periplasmic enzyme known to be required for resistance to copper ions. CueO from S . Typhimurium was expressed as a recombinant protein in Escherichia coli , and the purified protein exhibited a high cuprous oxidase activity. We have characterized an S . Typhimurium cueO mutant and confirmed that it is more sensitive to copper ions. Using a murine model of infection, it was observed that the cueO mutant was significantly attenuated, as indicated by reduced recovery of bacteria from liver and spleen, although there was no significant difference in recovery from Peyer's patches and mesenteric lymph nodes. However, the intracellular survival of the cueO mutant in unprimed or gamma-interferon-primed murine macrophages was not statistically different from that of wild-type Salmonella , suggesting that additional host factors are involved in clearance of the cueO mutant. Unlike a cueO mutant from E. coli , the S . Typhimurium cueO mutant did not show greater sensitivity to hydrogen peroxide and its sensitivity to copper ions was not affected by siderophores. Similarly, the S . Typhimurium cueO mutant was not rescued from copper ion toxicity by addition of the branched-chain amino acids and leucine.