Distamycin-based tetrapeptide (1) was covalently tethered to both ends of the central dihydroxyazobenzene moiety at either the 2,2 or 4,4 positions. This afforded two isomeric, distamycin-azobenzene-distamycin systems, 2 (para) and 3 (ortho), both of them being photoisomerizable. Illumination of these conjugates in solution at approximately 360 nm induced photoisomerization and the time course of the process was followed by UV/Vis and 1H NMR spectroscopy. The kinetics of the thermal reversion at various temperatures of cis to trans isomers of the conjugates obtained after photoillumination were also examined. This afforded the respective thermal-activation parameters. Both the molecular architecture and the location of the substituent around the core azobenzene determined the rate and activation-energy barrier for the cis-to-trans back-isomerization of these conjugates in solution. Duplex-DNA binding of the conjugates and the changes in DNA-binding efficiency upon photoisomerization was also examined by CD spectroscopy, thermal denaturation studies, and a Hoechst displacement assay. The conjugate 2 showed higher DNA-binding affinity and a greater change in the DNA-binding efficiency upon photoisomerization compared with its 2,2-disubstituted counterpart. The experimental findings were substantiated by using molecular-docking studies involving each conjugate with a model duplex d[(GC(AT)10CG)]2 DNA molecule