Leukemia inhibitory factor (LIF) maintains embryonic stem cells in an undifferentiated state. To establish stable cultures of zebrafish embryonic stem cells, recombinant zebrafish LIF (zLIF) is needed because the LIF sequence varies greatly between species. In the current study, we identified the zebrafish (Danio rerio) and pufferfish (Tetraodon nigroviridis) orthologs of lif from genomic databases, and we isolated a cDNA encoding zLIF. Synteny analysis and comparison of sequences identified zebrafish and Tetraodon orthologs of human LIF. The cDNA for zLIF encoded a predicted 215-amino acid protein with a putative 32-amino acid signal peptide, two disulfide bonds, and two N-linked glycosylation sites. We found that transcription of zlif starts at the hatching period during embryogenesis and is present in the brain, visceral organs, bone, and skin.