In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri.