Members of the Ets family of transcription factors are key regulators controlling prolactin (PRL) gene expression. Utilizing a transient transfection approach and the GH4 rat pituitary cell line, we have shown that Ets- 1 acts synergistically with the pituitary-specific POU homeodomain transcription factor, Pit-1, to mediate basal and Ras-induced regulation of the proximal (-425) rat PRL (rPRL) promoter. Although the transient transfection approach has provided important information regarding rPRL proximal promoter regulation, the role of Ets factors in the regulation of the intact, endogenous PRL promoter has not been explored. To address this area of question, we created several clonal GH4 cell lines that stably express either dominant-negative Ets (dn-EtsZ) or dominant-active Ets (VP16 Ets) constructs and used these cell lines as a model system to analyze the role of Ets factors on endogenous PRL gene expression. Northern blot analysis of these cells showed that PRL mRNA levels were dramatically reduced, by an average of 80%, in the cell lines expressing dn-Ets compared to vector-only controls. Conversely, stable expression of the dominant-active VP16 Ets led to an average threefold increase in PRL mRNA. GH4 cells expressing dn-EtsZ displayed significantly lower levels of intracellular PRL protein content and greatly diminished secretion of PRL into the cell culture medium, compared to vector-only controls. Consistent with our previous observations, the mRNA levels for growth hormone were unaffected by either dn-EtsZ or VP16 Ets expression. Expression of dn-EtsZ reduced Pit-1 mRNA levels by about 30%; however, the intracellular levels of Pit-1 protein were unchanged. Taken together, these results verify and strengthen the view that Ets factors play a critical role in the regulation of endogenous PRL gene expression and PRL protein production.