To investigate whether dietary α-linolenic acid (ALA) content alters the effect of β-carotene on mammary carcinogenesis, we conducted a chemically induced mammary tumorigenesis experiment in rats randomly assigned to four nutritional groups (15 rats per group) varying in β-carotene supplementation and ALA content. Two oil formula-enriched diets (15%) were used: one with 6g ALA/kg diet in an essential fatty acids (EFA) ratio of linoleic acid:ALA of 5:1 w/w (EFA 5 diet), the other with 24g ALA/kg diet in an EFA ratio of 1:1 w/w (EFA 1 diet), both designed with a similar linoleic acid content. β-Carotene was either added (10mg/kg diet per d) or not added to these diets. β-Carotene supplementation led to decreased tumour incidence and tumour growth when added to the EFA 5 diet, whereas it had no effect when added to the EFA 1 diet. The decreased tumour growth did not result from an involvement of lipoperoxidation (tumour malondialdehyde content being similar between the groups) or from an inhibition of tumour cell proliferation (as there was an unchanged S phase fraction in the tumours). We concluded that an adequate content of ALA in the diet is required to allow a protective effect of β-carotene in mammary carcinogenesis. Whether such an interaction between ALA and β-carotene influences the risk of breast cancer in women needs to be investigated.