OTC drugs have an important role in self-medication. However, the pharmacokinetic properties of some OTC drugs have not been fully investigated and reports concerning their drug interactions are insufficient. Several gastrointestinal drugs are available as OTC drugs. Because of their pharmacological properties, these drugs are often used concomitantly with other drugs. Therefore, it is important to predict the possible drug interactions among these drugs. In the current study, we investigated the inhibitory effects of five gastrointestinal drugs, namely loperamide, oxethazaine, papaverine, pirenzepine, and trimebutine, on CYP activities in human liver microsomes. Furthermore, we calculated the ratio of the intrinsic clearance of each CYP substrate in the presence or absence of the gastrointestinal drugs. The possibility of drug interactions in vivo was predicted by cut-off criteria. CYP3A4 activity was markedly inhibited by trimebutine, papaverine, and oxethazaine. Their inhibitory properties were competitive and the K-i values were 6.56, 12.8, and 3.08 mu M, respectively. Alternative R values of CYP3A4 exceeded the cut-off level. These results suggested that drug interactions mediated by CYP3A4 may occur during treatment with these gastrointestinal drugs, necessitating the confirmation of the clinical significance of these drug interactions to prevent unexpected adverse effects.