Uterine leiomyomas, which are myometrial smooth muscle tumors, secrete PRL. We investigated the actions of several hormones known to stimulate PRL secretion by the pituitary gland or decidua on PRL secretion by leiomyoma-derived smooth muscle cells (SMC) in monolayer culture. Cultures were verified to be SMC by immunostaining for smooth muscle alpha-action and desmin. Hormone treatments were performed in serum-free medium for 72 h. Medium was harvested every 24 h and assayed for PRL. 17 beta-Estradiol, progesterone, TRH, insulin-like growth factor-I, epidermal growth factor, and the GnRH agonist leuprolide did not affect PRL secretion by these SMC. Insulin caused a significant suppression of PRL secretion by 72 h, and this was accompanied by a 64% increase in total cell protein per well, which represented an increase in cell number. Cells were also plated at various densities to determine the effects of cell number on PRL secretion. The amount of PRL secreted per 1000 cells decreased significantly as cell number per well increased. Northern blot analysis identified PRL mRNA in fresh leiomyoma tissue. PRL mRNA in three independent cultures of SMC was then detected by reverse transcription and the polymerase chain reaction. Hybridization occurred only with the expected band of approximately 423 basepairs in size. We conclude that leiomyomas express PRL mRNA in vivo and that leiomyoma-derived SMC in culture continue to express the PRL mRNA and secrete PRL in the absence of ovarian steroids. PRL secretion by SMC in culture appears to be modulated primarily by changes in cell density.