We have cloned the zebrafish ortholog of the mammalian cyp26b1 gene. The predicted zebrafish cyp26b1 protein shares greater than 73% identity with mammalian homologues. cDNA transfection assays showed that like human cyp26b1, zebrafish cyp26b1 is involved in limiting the activity of retinoic acid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of embryonic RNAs suggested that no maternal cyp26b1 message is detectable. Zygotic cyp26b1 message could be detected at 75% epiboly by RT-PCR and localized to presumptive rhombomere 3 and rhombomere 4 at the early two-somite (2S) stage (10.5 hpf: hour post fertilization) by whole mount in situ hybridization. As development proceeds expression expands anteriorly to include rhombomere 2 at the 10S stage (14hpf). By 14S (16hpf) expression in the hindbrain has also expanded posteriorly and encompasses rhombomere 2 through rhombomere 6. At later stages, 24 through 48 hpf, additional expression was found in the eyes, diencephalon, midbrain-hindbrain boundary, cerebellum, pectoral fin and the pharyngeal arch primordia.