We evaluated the DNA damaging effects of ethanol and its major metabolite, acetaldehyde. Freshly isolated human lymphocytes from two healthy donors were incubated with 0, 1.56, 6.25, 25, and 100 mM of ethanol or acetaldehyde in complete medium for 1 h. We used a newly developed, sensitive, alkaline microgel electrophoresis technique to quantitate single- and double-strand DNA breaks or alkali-labile sites in individual cells. Ethanol did not induce DNA strand breaks. However, acetaldehyde induced both single-strand (even at the lowest concentration of 1.56 mM) and double-strand breaks (only at the highest concentration of 100 mM). Following exposure to acetaldehyde, cells were incubated in complete medium for 30, 60, and 120 min. During this incubation period, most cells were unable to repair DNA single- and double-strand breaks caused by acetaldehyde. We also observed a significant cell loss after exposure to acetaldehyde. To our knowledge, this is the first study demonstrating DNA single- and double-strand breaks by acetaldehyde.