The role of protein kinase AII (PKA II) in spatial memory retention in male rats and its regulation of cholinergic gene expression were explored through the effects of intrahippocampal infusion of H-89, a selective PKA II inhibitor. Alterations in escape latency, travel distance, and swimming speed in a Morris water maze were measured. Animals were trained for 3 days; each day included two blocks, and each block contained four trials. Stereotaxic surgery was employed for the infusions after the last trial on the third day of training, and the animals were tested 48 hr after surgery. Bilateral intrahippocampal infusion of H-89 (2.5 or 5 microM) into the CA1 region generated significant alterations in escape latency and traveled distance but not swimming speed. The response was fairly dose dependent, and the maximal effect was obtained with 5 microM H-89. After behavioral testing, several of the infused animals were transcardially perfused and their brains removed. Brain tissue sections from these rats were subjected to immunohistochemical staining analysis with anticholine acetyltransferase (ChAT) antibodies. These analyses indicated that 5 microM H-89 infusions qualitatively reduced the density of ChAT-containing cholinergic nerve terminals in the dorsal hippocampus. The intrahippocampal infusions with 5 microM H-89 also caused an apparent reduction in the number of ChAT-containing neurons in the medial septum. Our results suggest that PKA II is involved in regulation of cholinergic gene expression and plays an important role in spatial memory retention in rats.