The effect of cAMP on secretogranin II (SgII) gene transcription in GH₄C₁ (GH) cells is not observed unless protein synthesis is inhibited. We have defined elements in the SgII promoter that mediate regulation by cycloheximide (CHX) and forskolin (FSK) and characterized the nuclear proteins that interact with them. GH cells were transfected with p2774Luc, p351Luc, p242Luc, and p223Luc containing 2,612, 189, 80, and 61 bp of the SgII promoter upstream of the luciferase gene, respectively. Treatment with CHX and FSK increased promoter activity 8- to 12-fold in cells transfected with p2774Luc, p351Luc, and p242Luc but had no effect in cells transfected with p223Luc. The same 19-bp element (80 to 62) mediates regulation by CHX alone, as CHX caused a 3.8-fold increase in activity in GH cells transfected with p242Luc but not p223Luc. Gel mobility shifts using sequences 84 to 53 resulted in three complexes, which contained cAMP response element-binding protein heterodimerized with cAMP response element modulator or activating transcription factor-1. No differences were observed in complex formation when cells were treated with either CHX, FSK, or CHX and FSK. Thus CHX affects the response to FSK in GH cells by inhibiting the synthesis of a protein, which does not itself interact with DNA or affect the binding of CRE-binding proteins with the SgII promoter, but likely interferes with the interaction of CRE-binding proteins with the general transcriptional machinery.