We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M₂ muscarinic receptor; the -subunit of the heterotrimeric G protein, G_i2; and the inward rectifying K⁺ channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of G_i2 promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the G_i2 promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the G_i2 promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative G_i2 SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the G_i2 SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of G_i2 promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of G_i2 promoter activity, suggesting that SREBP1 may play a role in the regulation of G_i2 expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.