Transient transfection studies using gonadotrope-derived, alpha T3-1 cells were used to determine the DNA sequences of the mouse glycoprotein hormone alpha-subunit gene that mediate the transcriptional response to gonadotropin releasing hormone (GnRH). The roles of phorbol esters and cyclic AMP in mediating the GnRH response were also investigated. The initial studies demonstrated that a construct containing approximately 500 base pairs of alpha-subunit flanking sequence was sufficient to mediate responses to a GnRH agonist (GnRHa), phorbol myristate acetate (PMA) and a cAMP analog. Responses to combinations of cAMP and GnRHa or cAMP and PMA were approximately additive, whereas the response to the combination of GnRHa and PMA was similar to that seen with either of the agents alone. Cotransfection studies with an expression vector for the heat-stable inhibitor of the cAMP-dependent protein kinase demonstrated that GnRHa and PMA responses are not dependent on the cAMP-dependent kinase. Deletion analysis indicated that sequences between -507 and -205 were involved in mediating responses to GnRHa and PMA. To determine if this region alone could support responses to these agents, the -507 to -205 region was linked to a minimal promoter and tested in transient transfections. The results demonstrated that this region supports responses to GnRHa, PMA, and cAMP. Clustered point mutations of this region were used to further characterize sequences involved in the GnRH response. Mutations in two regions, one at positions -406 to -399 and one at positions -337 to -330, resulted in decreased responses to GnRH and PMA. There is no obvious sequence similarity between the two regions that are required for the GnRH response. An enhancer test demonstrated that multimers of the -416 to -385 region were able to function as a GnRH-responsive element when linked to a minimal promoter, although a single copy of this region was not sufficient to permit a response to GnRH. In contrast, multimers of the -344 to -300 region did not permit a response to GnRH, but enhanced basal transcription. These findings are consistent with the identification of a two-component GnRH response unit, which probably involves the functional cooperation of two different transcription factors. The observation that GnRH responsiveness appears to co-localize with PMA responsiveness suggests that GnRH effects on the alpha-subunit transcription are likely mediated by the protein kinase C pathway.