Objective The development of gallstones is influenced by obesity and alcohol. This study aimed to precisely quantify these risks and investigate whether the aetiological mechanism may involve serum lipids, for the first time using a European rospective cohort study. Methods The European Prospective Investigation into Cancer-Norfolk, recruited 25 639 men and women, aged 40 to 74 years, between 1993 and 1997. At enrolment weight, height and alcohol intake were recorded and nonfasting blood samples taken to measure serum triglycerides, cholesterol, high-density lipoproteins and low-density lipoproteins. The cohort was monitored for 14 years for symptomatic gallstones. Cox regression estimated sexspecific hazard ratios (HRs) for symptomatic gallstones adjusted for covariates. Results Symptomatic gallstones developed in 296 people (67.9% women). For each additional unit of BMI, the HR in men was 1.08 [95% confidence interval (CI) = 1.02–1.14]; in women the HR was 1.08 (95% CI= 1.06–1.11). Every unit of alcohol consumed per week decreased risk in men by 3% (HR = 0.97, 95% CI= 0.95–0.99) with no effect in women. Serum triglycerides increased risk in men (highest vs. lowest quarter HR= 2.02, 95% CI= 1.03–3.98) and women (HR =2.43, 95% CI =1.52–3.90). Increased high-density lipoprotein was associated with a decreased risk in men (highest vs. lowest quarter HR = 0.22, 95% CI= 0.09–0.52) and women (HR = 0.55, 95% CI =0.36–0.85). No effects were found for serum cholesterol and low-density lipoprotein. Conclusion Obesity and alcohol influence gallstone formation, possibly in part through their effects on serum lipids. Reducing obesity may prevent gallstones in the population, as 38% of incident cases of gallstones were associated with a BMI of more than 25.