An expressed sequence tag screen of a macrophage activation factor and lipopolysaccharide (LPS) stimulated goldfish macrophage subtractive library generated several transcripts of a putative teleost homologue of the toll-like receptor (TLR) family. The full-length TLR cDNA was sequenced and is predicted to encode a type I transmembrane protein with an extracellular domain containing leucine rich repeats and a cytoplasmic tail encoding a toll/interleukin-1 receptor domain. These findings indicate that the gene identified is the first teleost homologue of the TLR family reported. Constitutive expression of TLR was observed in unstimulated macrophages and was also observed in goldfish spleen and kidney but not in heart and liver tissues. A significant up-regulation of the TLR mRNA in cultured macrophages following treatments with each of bacterial LPS, heat-killed Aeromonas salmonicida, and live Mycobacterium chelonei was observed after 3 and 6 h post-stimulation, though with different kinetics from each other. A relative decline in TLR expression was observed after 24 h, but expression levels were still higher than that of unstimulated cells. Thus pathogen-derived factors appear to differentially modulate the expression of TLR in goldfish macrophages, which undoubtedly contributes to the orchestration and/or induction of functional immune responses in fish.