The cholinergic gene locus is comprised of two genes, the choline acetyltransferase gene and the vesicular acetylcholine transporter gene. The vesicular acetylcholine transporter gene is located within the first intron of the choline acetyltransferase gene. This arrangement permits coordinate regulation of the locus. Protein kinase A regulates expression of the cholinergic gene locus in PC12 cells. This regulation was found to be dependent on the presence of a 21-bp DNA sequence known as the repressor element- (RE- 1)/neuron-restrictive silencer element(NRSE). Repressor element-I silencing transcription factor (REST)/ neuron-restrictive silencer factor (NRSF), which binds to the RE-I/NRSE, is a zinc finger containing transcriptional repressor that blocks the expression of many neuronal RE-I/NRSE containing genes in nonneuronal cells. However, REST/NRSF expression has also been observed in neurons as well as the PC 12 cell line used in these studies. REST/NRSF truncated isoforms were expressed in neuronal cells, suggesting they also function in regulating neuronal gene expression. A study of REST4, one of the REST/NRSF isoforms, suggests that it regulates transcription of the cholinergic gene locus by blocking the repressor activity of REST/NRSF. Protein kinase A regulation of the cholinergic gene locus in PC 12 cells can thus be attributed, at least in part, to increased synthesis of REST4, which in turn derepresses the repressor activity of REST/NRSF.