Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Out of a total of 4154 expressed sequence tags (ESTs) generated from 4 cDNA libraries, 10 genes with immune-relevant functional annotations were selected for quantitative reverse transcription - polymerase chain reaction (QPCR) studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as up-regulated by A. salmonicida included interleukin 1 (IL1 ), interleukin 8 (IL8), a small inducible cytokine (SCYA), interferon regulatory factor 1 (IRF1), ferritin heavy subunit (FTH), cathelicidin, and hepcidin. This study is the first report on large-scale discovery of bacteria-responsive genes in cod, and the first to demonstrate up-regulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that stimulation with the bacterial antigens caused changes in a wide range of biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. In addition, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Taken together, our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.