Milk lipids of lactating rabbit mammary epithelial cells were labelled in vitro during a 3-min pulse with 14C-Na butyrate. After 1 h of incubation in the presence or not of prolactin, the different classes of lipids, in the tissues and the incubating mediums, were separated by thin-layer chromatography. The radioactivity of spots corresponding to the different classes of lipids (compared with the standards) was counted by liquid scintillation. In the tissues, triglycerides were the most strongly labelled constituent (97.7% of the radioactivity was bound to triglycerides). In the medium, triglyceride radioactivity was less; only 70.9% was bound to the triglycerides. In the presence of prolactin (10 micrograms/ml) the distribution of radioactivity in the tissues was not modified but in the medium, secreted triglycerides were more radioactive than in the control medium. In the presence of protamine sulfate (100 micrograms/ml), an inhibitor of lipoprotein-lipase, radioactivity in the secreted triglycerides decreased less, suggesting that this decrease was due to hydrolysis by lipoprotein lipase, probably during the process of exocytosis. Morphological evaluation on electron micrographs of the number of intracellular and extracellular lipid globules showed that prolactin increased the number of extracellular lipid globules; this suggested a higher secretion. These results show that the radioactivity of neosynthesized lipid constituents was modified during the secretion of milk lipids. Prolactin, which increased the total labelled lipids secreted, had an effect on the distribution of the radioactivity of the different lipid classes.