Doublecortin-like protein kinase (DCLK), a Ser/Thr protein kinase predominantly expressed in brain and eyes, is believed to play crucial roles in neuronal functions. However, the regulatory mechanisms for DCLK activation and its physiological targets are still unknown. In the present study, we found that a deletion mutant consisting of the catalytic domain of zebrafish DCLK, zDCLK(377-677), exhibited the highest activity among various mutants. Since fully active zDCLK(377-677) showed essentially the same substrate specificity as wild-type zDCLK, we used it to search for physiological substrates of zDCLK. When a zebrafish brain extract was resolved by isoelectric focusing and then phosphorylated by zDCLK(377-677), a highly basic protein with a molecular mass of approximately 90 kDa was detected. This protein was identified as synapsin II by mass spectrometric analysis. Synapsin II was found to interact with the catalytic domain of zDCLK and was phosphorylated at Ser-9 and Ser-58. When synaptosomes were isolated from zebrafish brain, both synapsin II and zDCLK were found to coexist in this preparation. Furthermore, synapsin II in the synaptosomes was efficiently phosphorylated by zDCLK. These results suggest that zDCLK mediates its neuronal functions through phosphorylation of physiological substrates such as synapsin II.