Two toll-like receptors (TLR3 and TLR5) were identified from a catfish cDNA fry library based on sequence similarity to other vertebrate TLR genes. Expression (using real-time PCR) of TLR3 and TLR5 was measured for two strains of channel catfish in previously non-exposed fish 2, 5, 8, and 21 days after experimental Edwardsiella ictaluri challenge to determine if TLRs are associated with host response to E. ictaluri infection. Expression of TLR5 was higher than TLR3 (P<0.0001). TLR3 expression in kidney was elevated in Norris strain (P=0.480) and differed over time in spleen (P=0.0134). Fold induction of TLR5 compared to non-exposed fish increased on days 5 (Norris; 154.72+/-62.12 fold induction) and 8 (USDA103; 164.65+/-50.56) post-exposure in liver and was slightly increased on day 5 (Norris; 10.17+/-24.73, USDA103; 42.56+/-24.73) in kidney. Upregulation of TLR3 suggests a more widespread function in primitive fish. TLR5 was highly expressed in liver tissue, which may be due to macrophage aggregation during ESC infection. This suggests that toll-like receptors are an important component of the innate immune system of catfish.