In human brain, the sole alcohol dehydrogenase (ADH) present in significant quantity has been shown to be Class III (chi) ADH and this ADH is ineffective in generating potentially toxic and reactive acetaldehyde from ethanol at concentrations attainable in living brain tissue. We have extended this finding to show that Class I ADH potentially present is undetectable even when concentrated several hundred-fold. Purified Class III ADH from human brain is identical in its pattern of tryptic peptides and in other properties to Class III ADH from human liver. Immunohistochemical staining and western immunoblots using polyclonal antibodies reveal that Class III ADH is widely distributed in brian and most concentrated in the subependymal layer and perivascular areas. Class III ADH closely resembles omega-hydroxyfatty acid dehydrogenase and a possible role for the brain enzyme is in the oxidation of long chain fatty alcohols and omega-hydroxyfatty acids.