The effect of various steroid and peptide hormones on the rate of incorporation of (14C)acetate into fatty acids was studied in MCF-7 cells, a human breast cancer cell line in continuous tissue culture. Physiologic concentrations of insulin elicited an increase in the rate of precursor incorporation. Addition of human placental lactogen and dexamethasone with insulin caused no further increment. Other hormones, such as 17beta-estradiol and 15a-dihydrotestosterone, had no effect on the rate of net labeled-acetate incorporation, although they stimulated the rate of general macromolecular synthesis. The stimulation by insulin appeared to be independent of glucose concentration, since the effect was seen in the absence of glucose. The insulin-induced increase was observed as early as 1 h following hormone addition and appeared to be maximal by 4 h. Inhibitors of RNA and protein synthesis had no effect on the insulin-mediated stimulation of precursor incorporation into fatty acids. Thus, these human cell lines in long term tissue culture may be of value as a model system in studying the action of physiologic concentrations of insulin.