The genomic materials from some Bacillus subtilis bacteriophages are found to contain 5‐(hydroxymethyl)‐2'‐deoxyuridine in place of thymine. Phage‐encoded proteins such as transcription factor 1 specifically and preferentially bind to the minor grooves of these hmU‐containing DNA but not to thymine‐containing DNA. Data from electrophoretic mobility shift assays suggest that the inherent, localized flexibility of hmU‐DNA, which is sequence‐specific, is responsible for its discriminative binding. We discuss here, from the NMR‐derived structural point of view, how differential DNA flexibility can contribute to specific binding of TF1 to hmU‐DNA. © 2000 John Wiley & Sons, Inc. Biopoly 52: 57–63, 1999