We have previously reported the detection of a 37 kD liver protein-acetaldehyde adduct in rats fed alcohol chronically with the AIN'76 diet. It was surprising that only one liver protein-acetaldehyde adduct was found. In this report, we have tried to detect additional protein-acetaldehyde adducts by electroimmunotransblot with rabbit anti-hemocyanin-acetaldehyde adduct IgG and to further characterize the 37 kD liver protein-acetaldehyde adduct. Sensitivity of electroimmunotransblot increased 10- to 20-fold when alkaline phosphatase-linked antibody was used in place of horseradish peroxidase, but only one protein-acetaldehyde adduct band was detected in liver. Feeding rats the Lieber-DeCarli alcohol diet also did not produce more protein-acetaldehyde adduct bands in electroimmunotransblot. Addition of cyanamide, an aldehyde dehydrogenase inhibitor, to the AIN'76 alcohol diet greatly increased the intensity of the 37-kD protein-acetaldehyde adduct band on electroimmunotransblot but did not produce other bands. The 37 kD protein-acetaldehyde adduct decayed in vivo with a half-life of 4 days when alcohol was removed from the diet. The 37 kD protein-acetaldehyde adduct in liver is cytosolic. Its interaction with anti-hemocyanin-acetaldehyde adduct IgG was blocked by polylysine-acetaldehyde adduct and polytyrosine-acetaldehyde adduct. It could be removed by immunosorption with anti-hemocyanin-acetaldehyde adduct IgG-bound immunoresin. When immunoblotted with anti-alcohol dehydrogenase and anti-aldehyde dehydrogenase antibodies, the alcohol dehydrogenase and aldehyde dehydrogenase bands in liver of alcohol-fed rats showed identical intensities before and after immunosorption.(ABSTRACT TRUNCATED AT 250 WORDS)