The diagnosis of drug-induced hepatitis is usually based on clinical criteria, with emphasis on both the temporal relationship between drug intake and liver injury and the exclusion of alternative causes. In vitro tests of lymphocyte sensitization to drugs are considered to have a low sensitivity. We investigated the possibility of detecting lymphocyte reactivity to drugs in drug-induced hepatitis by analyzing the lymphocyte proliferative responses to ex-vivo drug or metabolite antigens to improve the sensitivity of the in vitro test. Lymphocyte proliferative responses to five different concentrations of the drug and to ex-vivo drug antigens (serum collected from normal subjects after the ingestion of the drugs) were analyzed in 25 patients with a clinical diagnosis of drug-induced hepatitis, 27 healthy subjects and 10 individuals with a recent exposure to the same drugs without development of adverse drug reactions. In seven of the 25 patients, lymphocyte reactivity to drugs was detected (28%). The use of sera collected from healthy volunteers after drug intake (ex-vivo drug antigens) and the addition of a prostaglandin inhibitor to the cultures allowed the detection of lymphocyte sensitization in seven additional cases, increasing the detection ability from 28% to 56%. We suggest that the use of ex-vivo drug antigens may represent a significant contribution to the identification of the drug involved in cases of drug-induced hepatitis.