Short peptides derived from chromosomal proteins have previously been proposed to bind specifically to the minor groove of A,T-rich DNA [for a review, see M. E. A. Churchill and A. A. Travers (1991) Trends Biochem. Sci. 16, 92-97]. Using NMR spectroscopy, we investigated the DNA binding of SPRKSPRK, which is one such A,T-specific motif. Under the conditions studied SPRKSPRK interacts only nonspecifically with d(CGCAAAAAAGGC).d(GCCTTTTTTGCG). The peptides TPKRPRGRPKK, PRGRPKK, and PRGRP derived from the non-histone chromosomal protein HMG-I/Y, however, bind specifically to the central A,T sites of d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2. 2D NOE measurements show that the RGR segment of each peptide is in contact with the minor groove. The arginine side chains and the peptide backbone are buried deep in the minor groove, in a fashion generally similar to the antibiotic netropsin. Under the same conditions the peptide PKGKP does not interact with the same oligonucleotide duplexes, indicating that the arginine guanidinium groups are major determinants of the A,T specificity.