Density and pattern of nerve fibers with neuropeptide Y-like immunoreactivity (NPY-LI) and substance P-like immunoreactivity (SP-LI) in the rat dura mater encephali were investigated by light and electron microscopy using whole-mount preparations. NPY-LI fibers are observed throughout the encephalic dura mater. A remarkable net of NPY-LI nerve fibers is located in the walls of the sagittal and transverse sinuses. Beyond that NPY-LI network, distinct NPY-LI nerve fibers or plexus occur in the rostral falx, parietal dura mater of the olfactory bulb, supratentorial dura mater, parietal dura mater of the cerebellum, tentorium cerebelli and the ventral dura mater. Electron microscopic studies reveal that NPY-LI is exclusively located in unmyelinated axons of small and large nerve fiber bundles, with or without a perineural sheath. Immunopositive C-fibers are predominantly associated with the vascular bed. SP-LI nerve fibers have a moderate and more uniform distribution in the encephalic dura mater. A distinct plexus of SP-LI fibers follows the branches of the middle meningeal artery and the adjacent dura mater. SP-LI fibers are most prominent in the parietal dura mater of the cerebellum. Fine beaded SP-LI fibers, arising from larger SP-LI fiber bundles, are observed in close association to the capillary bed. SP-LI axons are all unmyelinated. They are found in larger nerve fiber bundles with a perineural sheath or in Schwann cells lacking any perineural sheath. The function of NPY-LI and SP-LI nerve fibers in the rat dura mater is discussed in relation to their topography, density and termination.