Cellular signaling events by which prolactin (PRL) might regulate gene expression were analyzed in rat ovarian tissues. Whole cell extracts (WCE) were prepared from ovaries of pregnant rats (Days 4, 7, 9-11, 15, 17, and 21) and of hormonally primed (estradiol and FSH) hypophysectomized (H) immature rats before, or 15 min to 24 h after, acute administration of PRL (10 micrograms, i.v.). The DNA binding activity in the WCE was analyzed by electrophoretic mobility shift assays using the alpha 2-macroglobulin (alpha 2M) promoter interleukin (IL)-6 response element (IL-6RE) known to confer PRL and IL-6 inducibility to transgenes in target cells, including cultures of luteinized granulosa cells. Injections of PRL stimulated the appearance of a specific binding activity in ovarian extracts of H rats and in corpora lutea and interstitial extracts of pregnant rats from Days 4-9 of pregnancy. The presence of this protein/DNA complex was transient. The greater amount of binding was observed in luteinized tissue and interstitial tissue compared to follicles; and the binding activity contained specific tyrosine phosphorylated Stat (signal transduction and activators of transcription) factors identified by specific antibodies as acute phase response factor (APRF or Stat 3) and mammary gland factor (MGF, or Stat 5 [a and b]). In contrast to the transient activation and appearance of these factors in response to acute PRL treatment as administered to H rats or to pulsatile PRL release as occurs in early pregnancy, elevated levels of the same activated Stat factors were observed in WCE of CL and interstitial tissue prepared at mid-gestation (Days 10-17) when endogenous release of rat placental lactogen (rPL) is chronically elevated in serum. During this period, administration of additional exogenous PRL did not stimulate further activation (binding) of the Stat factors. During luteal regression (Day 21 of gestation) no binding was observed in the absence of PRL, and the response to PRL was markedly diminished despite the constitutive presence of Stat proteins and the Janus kinase that phosphorylates and activates these factors. Elevated binding of these factors to the IL-6RE of the alpha 2M promoter was associated with the expression of alpha 2M mRNA in luteinized granulosa cells and corpora lutea, indicating that activation of Stat factors is one mechanism by which PRL/rPL transactivates the alpha 2M gene in the tissue.