The gene structure for S-100 beta subunit has been elucidated. The gene spans about 8 kbp and consists of 3 exons and 2 introns. The transcription initiation site was determined by an S1 nuclease mapping. The promoter region contains TATA-box-like and CAAT-box-like sequences. To examine the activity of the promoter sequence, a transfection of pS100 beta-lacZ fused gene to the cultured cells was carried out. C6 glioma cells showed a positive expression of beta-galactosidase. Gene-deletion experiments suggested the functional importance of the DNA fragment (22 bp) containing TATA-box-like and CAAT-box-like sequences. A factor protein that binds to the 100 bp DNA fragment containing the promoter sequence was specifically detected in the rat brain nuclear extract.