Ca 2+/calmodulin-dependent protein kinase I-delta (CaMKIδ), a fourth isoform of CaMKI, is known to be expressed ubiquitously in various tissues. However, the functional roles of this enzyme in vivo have not been fully clarified yet. To investigate the biological significance of CaMKId during zebrafish embryogenesis, we cloned and characterized two splice variants of zebrafish CaMKIδ, zCaMKIδ-L (392 AA) and zCaMKIδ-S (368 AA). Although recombinant proteins expressed in Escherichia coli showed weak protein kinase activities, they were activated when phosphorylated by mouse Ca 2+/calmodulin-dependent protein kinase kinase alpha. Activated CaMKIδ significantly phosphorylated various proteins including CREB, histones, and myelin basic protein in a Ca 2+/CaM-dependent manner. In COS7 cells, transiently expressed zCaMKIδ-L and zCaMKIδ-S were localized in the cytoplasm as in case of mouse CaMKIδ. During zebrafish embryogenesis, zCaMKIδ-L and zCaMKIδ-S were detected in the embryos after 36 and 60 hpf, respectively, by Western blotting using specific antibodies. Western blotting analysis in adult zebrafish demonstrated wide expression of zCaMKIδ-L in the brain, eyes, ovary, and skeletal muscle. In contrast, zCaMKIδ-S specifically localized in the brain. The knockdown of the zCaMKIδ genes with morpholino-based antisense oligonucleotides resulted in an increase of abnormal embryos with small heads, and this phenotype was rescued by coinjection with recombinant zCaMKIδ but not with kinase-dead mutant. These results clearly show the significance of zCaMKIδ-L and zCaMKIδ-S during zebrafish embryogenesis.