S100 beta is a calcium-binding protein produced and secreted by glial cells in the central and peripheral nervous systems. S100 beta promotes neuronal differentiation and survival but may be detrimental to cells if overexpressed. The selective overproduction of S100 beta has been implicated in the progression of the neuropathological changes in Alzheimer's disease. In addition, at high concentrations, S100 beta stimulates toxic intracellular pathways in cultured cells. To begin to define the regulation of S100 beta expression, we characterized the human S100 beta promoter and mapped its upstream regulatory elements by using a luciferase reporter system. The functional S100 beta promoter was localized to a region -168/ +697 containing 168 bp upstream of the transcription initiation site of the gene. This minimal promoter was active in a variety of cell types, including those of glial, neuronal, and non-neural origin. The human S100 beta promoter activity is regulated by both positive and negative regulatory elements located upstream in the 5' flanking DNA regions. The regions -788/ -391 and -1012/ -788 contain strong positive, cell type-specific regulatory elements. Negative regulatory elements were mapped to the more distal -4437/ -1012 and -1012/ -788 regions of the gene. The -4437/ -1012 negative element suppressed promoter activity in all cell types examined, except C6 glioma cells. These data demonstrate that the expression of the human S100 beta gene is under complex transcriptional regulation that allows for precise control of the S100 beta level in the nervous system.