The CtpA protein from pathogenic Listeria monocytogenes, is a P-type adenosine triphosphatase involved in copper homeostasis. To establish a role in pathogenicity for CtpA, a mutant strain was constructed by insertion of an antibiotic resistance cartridge into the ctpA gene. This mutant was then compared to the wild-type in tissue culture invasion assays and mouse infection studies. Mutants in CtpA, were unaltered for intracellular growth in J774 and HeLa cell lines. However, recovery of mutants from tissue of infected mice was dramatically reduced compared with the wild-type, and a significant impairment in terms of in vivo persistence in mixed-infection competition experiments was observed. These results demonstrate the significance of CtpA in establishing an in vivo infection by L. monocytogenes, and highlight some inadequacies of in vitro tissue culture monolayer assays for determining invasion and intracellular growth of a pathogen.