Inositol lipid kinases generate polyphosphoinositides, important regulators of several cellular functions. We have recently cloned two distinct phosphatidylinositol (PI) 4-kinase enzymes, the 210-kDa PI4KIII and the 110-kDa PI4KIII, from bovine tissues. In the present study, the distribution of mRNAs encoding these two enzymes was analyzed by in situ hybridization histochemistry in the rat. PI4KIII was found predominantly expressed in the brain, with low expression in peripheral tissues. PI4KIII was more uniformly expressed being also present in various peripheral tissues. Within the brain, PI4KIII showed highest expression in the gray matter, especially in neurons of the olfactory bulb and the hippocampus, but also gave a signal in the white matter indicating its presence in glia. PI4KIII was highly expressed in neurons, but lacked a signal in the white matter and the choroid plexus. Both enzymes showed expression in the pigment layer and nuclear layers as well as in the ganglion cells of the retina. In a 17-day-old rat fetus, PI4KIII was found to be more widely distributed and PI4KIII was primarily expressed in neurons. These results indicate that PI4KIII is more widely expressed than PI4KIII, and that the two enzymes are probably coexpressed in many neurons. Such expression pattern and the conservation of these two proteins during evolution suggest their nonredundant functions in mammalian cells.