Recent reports have described increased levels of a fast-moving hemoglobin (Hb) fraction in alcoholic patients and formation in vitro of stable adducts between acetaldehyde and Hb as well as between acetaldehyde and albumin. In the present study, we have found that factors other than acetaldehyde concentration can influence the rate of stable adduct formation. HbAo was purified and its 2,3-diPglycerate removed by dialysis. Under anaerobic condition and at 37 degrees, acetaldehyde (5 microM) reacted with deoxyHbAo to form 0.26 +/- 0.02 (+/- SEM) nmol of stable adduct/149 nmol Hb in 2 hr. By comparison, acetaldehyde reacted more slowly with oxyHbAo and carbonylHbAo; the rates were 0.21 +/- 0.01 (p less than 0.001) and 0.18 +/- nmol/149 nmol Hb/2 hr (p less than 0.001), respectively. Pyridoxal 5'-phosphate (50-500 microM), under anaerobic condition, inhibited by 36-56 percent the irreversible binding of acetaldehyde to deoxyHbAo. Ascorbic acid (2.5-10 mM) increased by 31-46 percent (p less than 0.001) the irreversible binding of acetaldehyde to human serum albumin and by 8-10 percent (p less than 0.05) the irreversible reaction of acetaldehyde with serum proteins. We conclude that the nonenzymatic binding of acetaldehyde to Hb, human serum albumin and serum proteins is influenced by factors other than acetaldehyde concentration. These factors include oxygen tension, pyridoxal 5'-phosphate and ascorbic acid. Among these factors, oxygen tension may be the most important in vivo.