To develop a method for quantitative electron microscopic immunocytochemistry on neural tissue of CNS, we tested the extent to which ethanol treatment would improve the penetration of immunoreagents through vibratome sections fixed in high concentrations of glutaraldehyde without compromising ultrastructure. Transverse or sagittal vibratome sections (60-80 microns) of spinal cord perfused with 1% formaldehyde plus 1% or 2.5% glutaraldehyde were washed in 50% ethanol for 0-70 min and stained to reveal immunoreactivity for neuropeptide Y (NPY). Semi-thin (1 micron) or ultra-thin sections were used to assess the depth to which NPY nerve fibers in the dorsal horn were stained. Without ethanol washing, immunoreactive nerve fibers were visualized only in the surface 5-10 microns of transverse or sagittal vibratome sections. In transverse vibratome sections, NPY nerve fibers, which ran perpendicular to the cut surfaces of the sections, were entirely stained after a 30-min wash in 50% ethanol. The numbers of NPY-immunoreactive varicosities and synapses were comparable at the surfaces and in the centers of the vibratome sections. In sagittal sections, where NPY nerve fibers ran parallel to the cut surfaces, fibers in the centers of vibratome sections could not be labeled even after 70 min in 50% ethanol. Substance P- and enkephalin (Enk)-immunoreactive nerve fibers could also be completely stained in transverse sections of spinal cord or medulla oblongata after 30-min exposure to ethanol. Ethanol washing had no significant deleterious effects on ultrastructure, although the amount of cytoplasmic matrix in neurons decreased with increasing exposure. These results indicate that washing with 50% ethanol for at least 30 min allows immunoreagents to penetrate completely through nerve fibers fixed with high concentrations of glutaraldehyde, as long as the fibers have cut ends at both surfaces of a vibratome section. This technique makes possible quantitative electron microscopic immunocytochemical studies and is proving a useful tool for defining synaptic connections in the CNS.