The aim was to determine the relative role of each of the lactogenic hormones (insulin, prolactin and hydrocortisol) and their combinations in regulating elongation and desaturation of polyunsaturated fatty acids and subsequently on composition of cellular lipid compartments in mammary epithelia. Cultured cells of the mammary gland epithelial cell line HC11 were subjected to 48 h of hormonal treatment with different combinations of insulin, hydrocortisone and prolactin. Only the combination of all three hormones induced differentiation according to the marker β-casein gene expression. Inclusion of insulin in the treatment medium increased total fatty acid amount by 50% and increased the concentration of monounsaturated fatty acids by 12% while decreasing that of saturated fatty acids by 35%. Changes in the levels of fatty acids by chain length and saturation paralleled mRNA expression of the desaturases and elongases, whose expression levels were regulated again by inclusion of all three hormones in the treatment medium. Gene expression levels of the Δ6 desaturase and elongase 5 genes (Elovl 5) increased by approximately 1.5-fold, whereas expression of Elovl 4 decreased in the presence of all three hormones. Insulin was the main hormone inducing compositional differences in membrane lipids, increasing phosphatidylethanolamine and phosphatidylinositol and decreasing sphingomyelin and cholesterol. The results indicate that mammary gland epithelial cells express five out of the seven known elongase subtypes which are regulated primarily by the processes of differentiation and produce major compositional changes in mammary gland epithelial cells.