It is generally accepted that cholesterol affects dynamic membrane properties and the function of membrane bound proteins involved in secretion processes. In the present study we employed GH4C1 cells treated with human lipoprotein-deficient serum (h-LPDS), exogenous cholesterol and high density lipoprotein (HDL3) to investigate the role of cholesterol cell content on PRL and GH basal release. Incubation of GH4C1 cells with h-LPDS decreased free cholesterol content and cholesterol added to the media increased it. HDL3 did not act as a cholesterol acceptor in either cholesterol-depleted or cholesterol-loaded cells; however, in depleted cells HDL3 was a net donor, significantly increasing cell cholesterol. Control or cholesterol loaded cells incubated in media with h-LPDS increased their secretion of PRL in parallel with the loss of cell cholesterol. Conversely, the addition of either cholesterol or HDL3 to cholesterol depleted cells inhibited PRL release. However, GH secretion was not modified by changes in free cholesterol in any of these situations. In the experiments in which HDL3 was present, a highly positive correlation was found between cholesterol cell content at the end of the experiment and PRL secretion, no effect could be related to the amount of added HDL3, suggesting that the HDL3 had no specific effect on the secretion of PRL or GH. Our results indicate that cholesterol cell content is an important factor in the release of PRL but not of GH, and emphasize the differences in the basal regulation of the secretion of both hormones.