In an attempt to characterize the ras signaling pathway, we studied the effects of expression vectors encoding the valine 12 mutant ras oncogene on rat prolactin (rPRL) promoter activity. Using this approach we have been able to dissect the interplay between the ras and the protein kinase A (PKA) pathways as they relate to neuroendocrine gene activation. Here we show that the ras oncogene product induces rPRL promoter activity selectively from 5- to 14-fold in GH4 rat pituitary tumor cells, whereas it has a minimal effect on the SV40 early promoter and no effect on the Rous sarcoma virus (RSV) or rat growth hormone promoters. By contrast, an inactivated form of ras (N-17 ras) did not stimulate the rPRL promoter, but rather inhibited it to 40% of control. Of note, activation of the PKA pathway by two different methods decreased the fold activation mediated by ras by at least 50%, whereas inhibition of the PKA pathway accentuated ras activation of the rPRL promoter. Although rPRL promoter activity is consistently induced by PKA activation in control GH4 cells, acute ras oncogene expression inhibited forskolin induction of rPRL promoter activity. Moreover, this ras-mediated interference of the forskolin activation of rPRL promoter activity was also noted in GH4 cells stably expressing ras. Taken together, these data show that the valine 12 ras oncogene activates the rPRL promoter selectively and, more importantly, that the ras and PKA signaling pathways are mutually antagonistic with respect to specific transcriptional activation of a neuroendocrine gene.