Mammalian cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase mainly expressed in the central nervous system and believed to be involved in neuronal functions. However, the functions of CDKL5 in fishes have not been investigated. Therefore, in this study, we cloned and characterized zebrafish CDKL5 (zCDKL5) and its substrate, amphiphysin 1 (zAmphi). Two alternative splice variants of zCDKL5, zCDKL5-Long (zCDKL5-L) and zCDKL5-Short (zCDKL5-5), and three splice variants of zAmphi, zAmph1(a), zAmph1(b) and zAmph1(c), were cloned from a zebrafish cDNA library. Using zAmph1(a) point mutants, we identified Ser-285 and Ser-293 as phosphorylation sites of zAmph1(a) by CDKL5. Transiently expressed zCDKL5-L and zCDKL5-5 colocalized with zAmph1(a) in the cytoplasm of 293T cells. RT-PCR analysis revealed that zCDKL5-L was first observed 12 hours post-fertilization (hpf) and increased thereafter, while zCDKL5-S appeared just after fertilization. zAmph1(a) was detected in all embryogenic stages and zAmph1(b) appeared from 12 hpf, but the expression of zAmphi, was not observed in our experiments. In adult fish, zCDKL5-L was mainly expressed in the brain, but zCDKL5-S showed ubiquitous expression. zAmphi a was observed most abundantly in the eyes, whereas zAmph1(b) was predominantly expressed in the brain. zAmph1(c) was scarcely detected. These results suggest that phosphorylation of Amph1 by CDKL5 may be a common feature throughout animal species. (C) 2016 Elsevier B.V. All rights reserved.