Previous studies have found an inverse relation between alcohol use and clinical gallstone disease, suggestive of a protective effect of alcohol use. However, such an inverse relation may (at least partly) be explained by a reduction of alcohol use because of symptoms related to clinical gallstone disease (protopathic bias). We empirically evaluated the consequences of different designs for the avoidance of such bias in a series of case-control studies. A first study deliberately used a design that is commonly seen in the literature. Cases of clinical gallstone disease, referred to hospital because of symptoms, were contrasted with general population controls. The results suggested an inverse relation between alcohol use and gallstones. Next, three alternative case-control studies were performed using designs that safeguard against protopathic bias. In none of these studies was an association between alcohol use and gallstones found. This demonstrates the probable existence of protopathic bias in case-control studies on alcohol use and gallstones. It is argued that earlier non-experimental studies on this topic were susceptible to such bias. This has most likely led to an overestimation of the protective effect.