The acyl-Coenzyme A oxidase 1, palmitoyl (ACOX1) catalyzes the first, rate-limiting step in peroxisomal -oxidation of medium- to very long straight-chain fatty acids. Zebrafish (Danio rerio) acox1 was characterized and compared with homologs from other sequenced genomes, revealing a remarkable conservation of structure in the vertebrate lineage. Strictly conserved regions of the deduced proteins included acyl-CoA oxidase and FAD binding domains, as well as a C-terminal peroxisomal targeting signal. Whole-mount in situ hybridization showed that zebrafish acox1 transcripts were diffusely distributed in early-stage embryonic cells, then discreetly expressed in the brain and widely present in the liver and intestine at later stages. An evolutionarily conserved alternative splicing of the corresponding acox1 primary transcript was identified in teleosts and tetrapods including mammals, giving rise, after exon skipping, to two splice variants, ACOX1-3I and ACOX1-3II. Real-time quantitative RT-PCR on zebrafish adult tissues indicated high levels of both variants in the liver, anterior intestine, and to a lesser extent, in the brain. However, the ACOX1-3II transcript variant was expressed seven times more in zebrafish brain than the ACOX1-3I variant. These data suggest a tissue-specific modulation of ACOX1 activity by exchanging exon 3 duplicated isoforms containing amino acid sequences that are potentially implicated in fatty acyl chain specificity. In addition, a significant pre-translational up-regulation of zebrafish and rainbow trout (Oncorhynchus mykiss) acox1 expression was observed in the anterior intestine after feeding. Taken together, these data indicate that ACOX1 alternative splicing isoforms play a key conserved role in the vertebrate fatty acid metabolism.