Objective: To investigate the relation between maternal smoking habits, plasma lipids and milk fatty acid (FA) content and composition. Design: Breastfeeding mothers who gave birth to healthy, full-term infants were recruited. Mothers were interviewed on smoking habits, being defined smokers (S) when usually smoking at least five cigarettes per day before pregnancy. Setting: Department of Pediatrics, San Paolo Hospital, Milan, Italy. Subjects: In total, 92 mothers: 61 non-S (NS) and 31 S. Interventions: Pooled hindmilk was collected at the first raise of milk (colostrum stage), 1, 3 and 6 months, and total lipid (TL) content and fatty acid (FA) composition were evaluated. Maternal dietary habits were assessed by a food-frequency questionnaire. Two subsamples (16 NS, 6 S) were investigated after delivery and at 3 months for serum lipids and FA status. At 6 months after delivery, the number of mothers still breastfeeding decreased to 30. Variables were compared using nonparametric tests. Results: In smoking mothers serum levels of triglycerides, cholesterol and low-density lipoproteins were higher, while those of high-density lipoproteins were lower. TL content in breast milk was similar in the two groups just after delivery but higher in milk from NS at 1 month. TL content and FA absolute amounts of linoleic, arachidonic, a-linolenic and docosahexaenoic (DHA) acid in breast milk were lower in S vs NS 1 month after delivery. Also 3 months after delivery, the breast milk of smoking mothers contained less DHA than the breast milk of nonsmoking mothers. Conclusions: Maternal cigarette smoking in early pregnancy is associated with higher plasma lipid levels and lower milk TL and DHA content in the first months of lactation.