Proliferative diabetic retinopathy is an important cause of visual impairment. We investigated whether the polymorphism of the β3-adrenoreceptor (β3-AR) gene, which is associated with insulin resistance and an earlier onset of NIDDM, was associated with proliferative diabetic retinopathy (PDR) in 215 Japanese NIDDM patients with a duration of diabetes of >10 years. The polymorphism of the β3-AR gene was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. The Trp64Arg allele of the β3-AR gene was significantly more frequent in the NIDDM patients with PDR (P = 0.002), but not in those with non-PDR (P = 0.151), than in NIDDM patients without diabetic retinopathy. Those with the mutation had an earlier onset of diabetes, a longer duration of diabetes, and higher current and maximal BMI values, compared with those without the mutation. Moreover, this mutation was also associated with higher serum triglyceride and decreased HDL-cholesterol levels. When adjustment was made for age, age at diagnosis, duration of diabetes, current BMI, systolic blood pressure, HbA1c, and serum lipids in a multiple regression analysis, a significant association was found between the Trp64Arg allele and diabetic retinopathy (P = 0.039). The Arg/Arg or Arg/Trp genotype was significantly associated with PDR, compared with the Trp/Trp genotype, with an odds ratio of 2.55 (95% CI 1.25-5.16). We concluded that the β3-AR gene polymorphism is a newly identified risk factor for PDR.