In Nb2 node rat lymphoma cells, the effects of prolactin (PRL) on the rates of incorporation of several precursors into neutral lipids, phospholipids and proteins were determined. The onset of the PRL stimulation of radiolabeled-precursor incorporation into lipids occurred between 1 and 4 hours after PRL addition to Nb2 cells; precursors employed included [14C]-acetate, [3H]-glycerol, [32P]O4, [3H]-choline, [3H]-ethanolamine, [3H]-serine and [3H]-myoinositol. No effects were observed during the initial 60 min of culture with PRL. The effects on precursor incorporation that occur after 1 hr of PRL exposure are likely related to the stimulation of cell growth by PRL. In cells that were prelabeled with the radiolabeled precursors and subsequently incubated with PRL, PRL had no effect on the metabolism of the radiolabeled phospholipids or the accumulation of phospholipid products until several hours after hormone addition. We would conclude from these studies that the initial (60 min) effect of PRL on Nb2 node lymphoma cells does not likely use a signal transduction mechanism that involves products derived from the cellular phospholipids.