The antitumor antibiotic 7-aminoactinomycin D has been previously demonstrated to bind and stabilize transient hairpin structures formed by single-stranded DNA. Those experiments suggested that DNA secondary structures are, viable targets for development of novel antitumor or antiviral compounds. Interestingly, when 7-aminoactinomycin D binds to hairpins formed by selected single-stranded DNAs having a high affinity for the drug, the fluorescence quantum yield, average fluorescence lifetime, excitation wavelengths, and emission wavelengths for the drug are significantly different from those observed when it is free in solution or bound to duplex, B-form. DNA. This suggests a unique physical chemical environment within certain DNA hairpins that is specifically recognized by the drug. To understand the environmental contribution to the fluorescence properties of the bound drug, we used solvatochromism techniques, whereby 7-aminoactinomycin D fluorescence properties were determined in 20 solvents with known polarization (pi), hydrogen bond donor (alpha), and hydrogen bond acceptor (beta) properties. These data reveal solvent hydrogen bonding to 7-aminoactinomycin D in the excited state. Semiempirical molecular orbital methods have been used to calculate the electronic structure of the first singlet excited state of 7-aminoactinomycin D. The calculations indicate the redistribution of electron density in the excited-state involves the oxygen atom attached to carbon 3 of the phenoxazone ring system. Hence, the solvent-dependent changes in fluorescence properties of 7-aminoactinomycin D involve solvent hydrogen bonding to this oxygen. This oxygen is protected from solvent in the binding site for 7-aminoactinomycin D on selected DNA hairpin secondary structures due to the unique chemical environment of this site.