Bone sialoprotein (BSP) is a noncollagenous matrix glycoprotein localized predominantly in mineralized tissues but also detected in extraskeletal sites undergoing focal mineralization. We have previously characterized the human BSP gene and have shown that the upstream sequence contains inverted TATA and CCAAT motifs at the expected locations from the transcriptional start site (J. M. Kerr et al. ) and a potential YY1 binding motif located within the first 30 bp of intron 1 of the human gene. Deletion analyses of the human BSP promoter/exon 1 sequence fused to a CAT reporter gene indicate that CCAAT enhances basal transcription of BSP in transiently transfected rat UMR106-01 BSP osteosarcoma and rat skin fibroblasts. Though this enhancing activity was lost with inclusion of 68 bp of intron containing a YY1 motif in these constructs, reporter activity in the UMR106-01-BSP cells was elevated four- to seven-fold relative to that of rat fibroblasts. Gel electrophoretic mobility shift, UV-crosslinking, and south-western experiments indicate that YY1 is present only in the extracts of nuclei isolated from the UMR cells and may contribute to the elevated transcriptional activity of the human BSP promoter construct in UMR106-01-BSP.