Elevated mean corpuscular volume (MCV) is a traditional biological marker for alcohol abuse and alcoholism, but the underlying mechanism is unclear. Three recent epidemiologic studies consistently showed that MCV was elevated by alcohol drinking more markedly among individuals with genetically inactive aldehyde dehydrogenase-2 (ALDH2) (encoded by ALDH2*2 mutant allele) than those with active ALDH2 (encoded by ALDH2*1/2*1 genotype), suggesting that the elevated MCV was etiologically linked to acetaldehyde exposure. The purpose of the present study was to clarify further this relationship by examining the status of folate and vitamin B12. The study participants were 159 men who were aged 40 to 69 years and randomly selected from a Japanese rural population. The genetic polymorphism of ALDH2 was determined by PCR-restriction fragment length polymorphism method; data on alcohol drinking and other lifestyles were collected using a structured questionnaire; serum concentrations of folate and vitamin B12 were measured using the protein competitive reaction method, and blood cell counts were measured by routine methods. A multiple linear regression model was used to analyze the data. : The relationship between alcohol drinking and serum folate concentration was significantly different between ALDH2 genotypes, indicating that the reduction of serum folate by alcohol drinking was more marked in men with ALDH2*1/2*2 than those with ALDH2*1/2*1. The relationship between alcohol drinking and elevated MCV was significantly stronger in men with ALDH2*1/2*2 than those with ALDH2*1/2*1 even after adjustment for serum folate and vitamin B12 concentrations. These findings indicate that acetaldehyde plays a significant role in the development of decreased serum folate concentration and elevated MCV by alcohol drinking.