Maternal smoking during pregnancy has been associated with a reduced content of n-3 long-chain PUFA (LC-PUFA) in breast milk, thereby reducing the intake of key nutrients by the infants. We postulated that the mammary gland is affected by maternal smoking in the process of n-3 LC-PUFA secretion into milk. This prompted us to investigate the effects of cigarette smoke on the synthesis of n-3 LC-PUFA in vitro by using a line of healthy epithelial cells from the human mammary gland, MCF-10A. Cells were exposed to cigarette smoke under controlled conditions by adding to the medium aliquots of horse serum containing smoke components, as analyzed by GC-MS. The major findings concern the inhibition of both the conversion of the precursor 14C-ALA (alpha-linolenic acid) to n-3 LC-PUFA and of the A5 desaturation step (assessed by HPLC analysis with radiodetection of n-3 FAME) following exposure to minimal doses of smoke-enriched serum, and the dose-dependent relationship of these effects. The data indicate that exposure to cigarette smoke negatively affects the synthesis of n-3 LC-PUFA from the precursor in mammary gland cells.