Signature-tagged mutagenesis was used to identify genes essential for survival of Yersinia ruckeri in its natural host, rainbow trout, Oncorhynchus mykiss. A mini-Tn5-Km2 signature-tagged mutant, C6-1, was missing from rainbow trout kidney at 7 days after an immersion challenge. The transposon insertion in C6-1 was in a homologue of the znuA gene of Escherichia coli that encodes ZnuA, a zinc-binding periplasmic protein of the high-affinity zinc transporter ZnuABC. Further sequencing of the C6-1 locus in Y. ruckeri identified homologues of two other genes: znuB, encoding a putative inner membrane permease, and znuC, encoding a putative ATPase. When present on a low-copy plasmid, the znuABC locus of Y. ruckeri fully restored growth of a zinc transport-deficient DeltaznuABC mutant of E. coli. Unlike DeltaznuABC mutants of E. coli and Salmonella typhimurium, the DeltaznuABC mutant of Y. ruckeri did not demonstrate significantly slower growth in zinc-deficient M9 minimal medium or in Luria-Bertani (LB) medium supplemented with the metal chelators EDTA and tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). In LB medium, the znuA::lacZ and znuCB::lacZ transcriptional fusions of Y. ruckeri were derepressed by addition of EDTA and TPEN and were repressed by addition of zinc and manganese. In a competitive challenge by immersion, the DeltaznuABC mutant was unable to compete with the parental strain and survived poorly in rainbow trout kidney, indicating that the ZnuABC transporter has a role in establishing and maintaining a rainbow trout infection by Y. ruckeri.