Glucose and acetaldehyde react covalently with albumin to form the post‐translationally modified group of proteins, the glycosylated albumins and the acetaldehyde albumins, respectively. This study contrasts the binding ability of a major acetaldehyde albumin fraction synthesized in vitro with glycosylated albumin. A microdialysis rate method, using either [34C]monoacetyidiaminodiphenyl sulfone (MADDS), a deputy ligand for bilirubin, or [34C]diazepam, was employed to evaluate binding at these two sites. Our results indicate that prolonged exposure of purified human serum albumin to acetaldehyde results in a major acetaldehyde albumin fraction that lacks the ability to bind MADDS and diazepam. This fraction migrates identically to albumin on SDS polyacrylamide gel electrophoresis, but exhibits microheterogeneity with a more acidic pi band as seen on analytical isoelectric focusing. We suggest that altered drug binding in alcoholics may be partially explained by altered binding ability of acetaldehyde albumins.