The organization, sequence and transcriptional regulation of the murine S100{dollar}\beta{dollar} gene have been studied. The gene is approximately 9 kb in length and is composed of three exons and two introns. The murine S100{dollar}\beta{dollar} gene contains a TATA box (AATAA) and a reverse CCAAT box (ATTGG) located at 30 nucleotides and 92 nucleotides upstream of the transcription start site, respectively. A 149 bp DNA fragment ({dollar}-{dollar}157/{dollar}-{dollar}9) spanning the TATA box and reverse CCAAT box functions as a promoter. The murine S100{dollar}\beta{dollar} promoter drives a 4-fold higher level of transcription in C6 rat glioma cells than in non-glial 3T3 murine fibroblast cells, suggesting the existence of a cell type-specific regulatory element within the promoter region. The 5{dollar}\sp\prime{dollar}-flanking region suppresses transcription from the homologous S100{dollar}\beta{dollar} as well as heterologous SV40 promoters in an orientation-independent fashion. However, the 5{dollar}\sp\prime{dollar}-flanking region exhibits cell-type specificity when suppressing the S100{dollar}\beta{dollar} promoter-dependent transcription, indicating its involvement in the regulation of the cell type-specific expression of the murine S100{dollar}\beta{dollar} gene. In order to map the cell type-specific regulatory elements, transcriptional analyses of various deletions of the 5{dollar}\sp\prime{dollar}-flanking region were carried out in both C6 and 3T3 cells. Two cell type-specific negative regulatory elements, one active in non-glial cells and another active in glial cells, were mapped to the regions {dollar}-{dollar}1552/{dollar}-{dollar}1234 and {dollar}-{dollar}1234/{dollar}-{dollar}551, respectively. A strong negative regulatory element and a relatively weak negative regulatory element were located in the regions {dollar}-{dollar}551/{dollar}-{dollar}157 and {dollar}-{dollar}1669/{dollar}-{dollar}1552, respectively. These results lead to the conclusion that the murine S100{dollar}\beta{dollar} gene is under complex transcriptional regulation involving tonic negative control exerted by a combination of multiple cis-acting regulatory elements including cell type-specific elements. Gel mobility shift assay with the cell type-specific regulatory element III (CSREIII, {dollar}-{dollar}1552/{dollar}-{dollar}1234) showed that a unique trans-acting protein factor may be present in 3T3 nuclear extract but absent in C6 nuclear extract. This factor can specifically bind to a 17 bp DNA fragment ({dollar}-{dollar}1513/{dollar}-{dollar}1497) which contains a consensus silencer element. These findings suggest that the glial-specific expression of the murine S100{dollar}\beta{dollar} gene is probably mediated by both cis-acting silencer elements and trans-acting protein factors.