We evaluated three markers of ethanol intake [whole blood associated acetaldehyde (WBAA), serum beta-hexosaminidase, and gamma-glutamyl transpeptidase (GGT)] in four groups of subjects: teetotalers (n = 104), random insurance applicants or "normals" (n = 1,010), subjects enrolling in an alcohol treatment program or "alcoholics" (n = 31), and subjects attending outpatient drug/alcohol treatment follow-up clinics (n = 128). Significant differences (p < 0.004 for each assay and each comparison) were found in the mean values between teetotalers and normals and normals and alcoholics. Male teetotalers and normals had significantly (p < 0.002) higher levels of WBAA than females of the same group. Male normals had significantly higher levels of GGT than females (p < 0.001). GGT increased with age in the normal population into the fifth decade and decreased thereafter. WBAA was the most sensitive assay with 97% of alcoholics having values above the 99th percentile for the teetotaler population (vs. 66% for serum beta-hexosaminidase and 70% for GGT). None of the alcoholic subjects had values for all three assays below the 99th percentile for teetotalers compared with 21% of those in follow-up and 72% of normals. We conclude that WBAA appears to be the best of the three markers studied and that measurement of multiple markers for ethanol use appears clinically useful and incremental.