Obesity is considered to be an important risk factor for the formation of gallstones. The relationship is well established for women but not for men. In a long-term follow-up study of middle-aged men the relationship between various markers of obesity and the incidence of clinically diagnosed gallstones during 25 years of follow-up was studied. Information on the presence of gallstones was obtained by self-report and verified through medical records after death. Of the 860 men who were between 40 and 59 years old at the start of the study, 54 developed gallstones, yielding an incidence rate of 3.1/1000 person-years. Cox proportional hazard models were used to examine the associations between the risk factors and newly diagnosed gallstones. Univariate analysis revealed that the subscapular-to-triceps skinfold thickness ratio (STR) yields a significant positive association (HR upper quartile: 2.5, 95% CI: 1.1-5.7). Subscapular skinfold thickness had a borderline significant, positive association, which became significant after exclusion of subjects who developed symptomatic gallstones within the first 3 years of follow-up (HR upper quartile: 2.5, 95% CI: 1.0-6.2). The multivariate model revealed that the association of STR with clinically diagnosed gallstones was independent of Body Mass Index. Our results indicate that regional fat distribution, as measured by the subscapular-to-triceps skinfold thickness ratio, may play an important role in the formation of gallstones in men, as was previously found for women in other studies.