The presented results demonstrate that human SOX3 promoter possesses three CCAAT box control elements involved in the regulation of SOX3 gene expression in NT2/D1 cells. By mutational analysis we have shown that all three elements are of functional relevance for constitutive SOX3 expression. Electrophoretic mobility shift assays indicate that the active complexes at three sites involve the ubiquitously expressed CCAAT binding protein NF-Y. The involvement of NF-Y in the up-regulation of SOX3 expression in NT2/D1 cells was demonstrated in vivo by Northern and Western blot analyses. Furthermore, in co-transfection experiments we have shown that NF-Y mediates transcriptional activation of SOX3 promoter. Our data indicate that multiple CCAAT control elements are involved in the regulation of the SOX3 promoter, suggesting that NF-Y functions as a key regulator of SOX3 gene expression. Further, our results indicate that these elements can be recognized as modulators of retinoic acid induced activation of SOX3 expression.