Junctions between ssDNA and dsDNA sequences are important in many cellular processes, including DNA replication, transcription, recombination, and repair. Significant transient conformational fluctuations ("DNA breathing") can occur at these ssDNA-dsDNA junctions. The involvement of such breathing in the mechanisms of macromolecular complexes that operate at these loci is not well understood, in part because these fluctuations have been difficult to measure in a position-specific manner. To address this issue we constructed forked or primer-template DNA constructs with 1 or 2 adjacent 2-aminopurine (2-AP) nucleotide residues (adenine analogues) placed at specific positions on both sides of the ssDNA-dsDNA junction. Unlike canonical DNA bases, 2-AP absorbs, fluoresces, and displays CD spectra at wavelengths >300 nm, where other nucleic acid and protein components are transparent. We used CD and fluorescence spectra and acrylamide quenching of these probes to monitor the extent and nature of DNA breathing of A-T base pairs at specific positions around the ssDNA-dsDNA junction. As expected, spectroscopically measurable unwinding penetrates approximately 2 bp into the duplex region of these junctions under physiological conditions for the constructs examined. Surprisingly, we found that 2-AP bases at ssDNA sites directly adjacent to ssDNA-dsDNA junctions are significantly more unstacked than those at more distant ssDNA positions. These local and transient DNA conformations on both sides of ssDNA-dsDNA junctions may serve as specific interaction targets for enzymes that manipulate DNA in the processes of gene expression.