The interactions with DNA of tetrapeptide amides containing lysine a t the N-terminal position and aromatic amino acids a t the second and fourth positions (Ala a t position three), 1-6, have been investigated by nmr, CD, and viscometric methods. Tetrapeptides with N-terminal lysine and a single aromatic amino acid, 7-10, were investigated as controls. Significant decreases in DNA viscosity occurred on addition of 7, with the aromatic group a t the second position, but not with any of the other single aromatic amino acid peptides. All of the tetrapeptides with two aromatic groups caused DNA viscosity decreases which were two to three times larger than with 7. Peptides with p-nitrophenylalanine (p-NO,Phe) as the aromatic group were synthesized for nmr studies because of its simpler aromatic nmr spectrum relative t o Phe. Large upfield shifts of the aromatic proton signals were obtained when the amino acid in the second position was ~-p -N o ~P h e , and the fourth position contained either p-N0,Phe or Phe. Such peptides also caused the largest DNA viscosity decreases on complex formation. Smaller upfield shifts of the aromatic signals were obtained when the amino acid in the second position was L-Phe or a 1 1 isomer of Phe or p-N02Phe. With all peptides, larger upfield nmr shifts were obtained with heat-denatured, recooled DNA than with native DNA under the same conditions. As with nmr, CD results are quite different for the peptides with L and D amino acids at the second position. All of the results can be interpreted in terms of a model in which lysine interacts stereospecifically with the backbone in a DNA double helix and the aromatic group at the second position stacks strongly with the base pairs when the amino acid is a n L isomer. The aromatic group a t the fourth position can also interact with the base pairs, but primarily through a sideways stacking of the aromatic group with base pairs for either or D isomers. Because of covalent constraints on the separation distance for the two aromatic groups in the tetrapeptides, they must stack on opposite sides of the same base pair in violation of the neighbor exclusion principle observed with classical intercalators. This stacking a t the same base pair no doubt accounts for the larger viscosity decreases in DNA with the peptides containing two aromatic groups relative to those with a single aromatic group. Such protein-induced conformational changes may be very important in protein-DNA or RNA recognition, and in opening of DNA by single-stranded binding proteins and enzymes such as RNA polymerase.