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Human centromere protein B (CENP-B) has a sequence-specific DNA binding activity. We previously reported several CENP-B binding motifs by analysing synthetic oligonucleotides as well as alphoid DNA isolated from the human genomic library. Here, we examined the size requirement and nucleotide specificity of human CENP-B binding sequences in vitro. We synthesized three sets of mixed oligonucleotides containing diverged authentic binding sites (CTTCGTTGGAAACGGGA) in which certain pairs of nucleotides (underlined) were degenerated. Each oligonucleotide with a defined sequence was separately introduced into a plasmid and mixed with GST-fused recombinant CENP-B. The DNA-protein complex formed was affinity purified with glutathione Sepharose. Any nucleotide substitutions at the positions 1, 2 and 17 did not significantly influence the recovery, while the substitutions at positions 3, 4 and 16 did, suggesting that the internal 14-bp motif (TCGTTGGAAACGGG) constituted the minimum requirement. However, it showed a lower affinity to CENP-B, compared with the authentic motif. The inclusion of T at the 5′ end greatly increased the affinity, and the further addition of A or T at the 3′ end (TTCGTTGGAAACGGGA/T) offered affinity similar to the authentic motif. The first nucleotide of the 17-bp authentic binding motif may not be essential for CENP-B binding.