Early neurogenesis and neuronal differentiation are precisely controlled by a series of genes. Sox3 is expressed in the brain from initial stages of development and it is considered to be one of the earliest markers in vertebrates, playing the role in specifying neuronal fate. In order to elucidate molecular mechanisms underlying the regulation of the human SOX3 gene expression, computer software was used to search the matrix database and to identify potential transcription binding sites within the human SOX3 promoter. Among the number of putative consensus binding sites, three evolutionary conserved CCAAT boxes, representing the putative binding sites for the general transcription factor NF-Y, were identified. By mutational analysis we tested the ability of the single, double and triple site mutants and its wild-type counterpart to drive expression of the cat reporter gene in stem and RA induced NT2/D1 cells. We have shown that all three motifs are of functional relevance for SOX3 expression. Performed EMSA and "supershift" experiments proved that NF-Y binds to all three CCAAT boxes. Results obtained in co-transfection experiments, using NF-Y expression vectors, demonstrated that NF-Y mediates transcriptional activation of the SOX3 promoter. Our results strongly suggest that all three CCAAT box motifs, NF-Y binding sites, present in the SOX3 promoter play functional role as transcriptional activators of this gene.