Indirect evidence from both epidemiological studies and animal experiments suggests that insulin may promote breast cancer development. In this study, we directly tested for a promoting effect of insulin on mammary carcinogenesis in Sprague-Dawley rats. Fifty day-old female rats received an i.p. injection of 37.5 mg/kg methylnitrosourea (MNU). Five days later, the animals were randomized into two groups. One group received insulin injections five times/week until the time of death, while the other control group received similar injections of normal saline. Over the course of 26 weeks following MNU treatment, the mammary tumour incidence in the insulin-treated group did not differ significantly from the saline-treated controls. Furthermore, the number of tumours per tumour-bearing rat did not differ between groups. Our results demonstrate that insulin is not a promoter of mammary carcinogenesis in this model.