The leukotriene B₄ (LTB₄) receptor 1 (BLT1) is a high affinity receptor for LTB₄, a chemotactic and inflammatory eicosanoid. The LTB₄ receptor 2 (BLT2) was originally identified as a low affinity receptor for LTB₄, and, more recently, as a high affinity receptor for 12-hydroxyheptadecatrienoic acid (12-HHT). The zebrafish BLT receptors have not been previously identified and the in vivo functions of these receptors have been unknown. In this paper, we describe one zebrafish BLT1-like receptor, Blt1, and two zebrafish BLT2-like receptors, Blt2a and Blt2b. Cells expressing Blt1 exhibited LTB₄-induced intracellular [Ca²⁺] increases, inhibition of cAMP production, ligand-dependent [³⁵S]GTPγS binding, and transforming growth factor-α (TGFα) shedding activity in a dose-dependent manner, similar to human BLT1. Cells expressing Blt2a and Blt2b exhibited 12-HHT- and LTB₄-induced intracellular [Ca²⁺] increases, inhibition of cAMP production, [³⁵S]GTPγS binding, and TGFα shedding activity, with a dose-dependency similar to human BLT2. Reverse transcription (RT)-PCR analysis and whole-mount in situ hybridization revealed that blt1, blt2a, blt2b, zebrafish LTA₄ hydrolase (lta4h), and zebrafish 5-lipoxiganase (5lo) are expressed in zebrafish embryos. Knockdown of blt1 by morpholino antisense oligonucleotides resulted in delayed epiboly at gastrulation. Consistently, knockdown of lta4h, an enzyme mediating LTB₄ production, induced a phenotype similar to knockdown of blt1. These results suggest that the LTB₄-BLT1 axis is involved in epiboly in zebrafish development.