This study investigated the role of hippocampal CaM-kinase II (calcium/calmodulin-dependent protein kinase II) in spatial learning. In Experiment I, three groups of rats received 1, 2 or 5 days of training on a spatial task in the Morris water maze with a hidden platform, while a control group was trained on a nonspatial task with a visible platform. The acquisition rate in the spatial task was slower than that in the nonspatial task. However, rats receiving 5 days of spatial training had the highest Ca(2+)-independent activity of CaM-kinase II compared with the controls receiving nonspatial training and rats having 1 or 2 days of spatial training. Furthermore, the level of hippocampal Ca2+-independent CaM-kinase II activity was correlated with the final performance on the spatial task. In Experiment II, rats received intra-hippocampal injections of a specific CaM-kinase II inhibitor-KN-62-before each training session. In comparison with the vehicle-injected controls, pretraining injection of KN-62 retarded acquisition in the spatial task but had no effect on the nonspatial task. These results, taken together, indicated that the activation of CaM-kinase II in the hippocampus is not only correlated to the degree of spatial training on the Morris water maze, but may also underlie the neural mechanism subserving spatial memory.