The distribution of tissue plasminogen activator (tPA) messenger RNA in rat brain was studied using in situ hybridization with 35S UTP-labeled RNA probes derived from a full-length tPA cDNA. Sense strand controls produced low, even backgrounds, with small elevations in the hippocampus. Full-length antisense probes produced strong signals over cerebral ventricular ependyma (including ependyma of the subcommissural organ), meninges, blood vessels, and Purkinje cell layer of the cerebellum, as well as strong signals over scattered cells throughout the brain. Some of these scattered labeled cells were large with lightly stained nuclei, while others were small with darkly stained nuclei. The large labeled cells, which were probably neurons, constituted 6% and 8% of cells in the brain stem and neocortex, respectively, and 100% of Purkinje cells. The small cells, which were present in all areas of the brain, constituted 3-11% of cells in individual brain areas.