Organisms have evolved to live in a variety of complex environments, which clearly has required cellular biology to accommodate to extreme conditions of hydraulic pressure and elevated temperature. In this work, we exploit single-molecule Forster resonance energy transfer (FRET) spectroscopy to probe structural changes in DNA hairpins as a function of pressure and temperature, which allows us to extract detailed thermodynamic information on changes in free energy (Delta G degrees), free volume (Delta V degrees), enthalpy (Delta H degrees), and entropy (Delta S degrees) associated with DNA loop formation and sequence-dependent stem hybridization. Specifically, time-correlated single-photon counting experiments on freely diffusing 40A DNA hairpin FRET constructs are performed in a 50 mu m x 50 mu m square quartz capillary cell pressurized from ambient pressure up to 3 kbar. By pressure-dependent van't Hoff analysis of the equilibrium constants, AV for hybridization of the DNA hairpin can be determined as a function of stem length (n(stem) = 7-10) with single base-pair resolution, which further motivates a simple linear deconstruction into additive stem (Delta V degrees(stem) = Delta V degrees(bp) x n(stem)) and loop (Delta V degrees(loop)) contributions. We find that increasing pressure destabilizes the DNA hairpin stem region [Delta V degrees(bp) = +1.98(16) cm(3)/(mol bp)], with additional positive free volume changes [Delta V degrees(loop) = +7.0(14) cm(3)/mol] we ascribe to bending and base stacking of the 40-dA loop. From a van't Hoff temperature-dependent analysis of the DNA 40A hairpin equilibria, the data support a similar additive loop/stem deconstruction of enthalpic (Delta H degrees = Delta H degrees(loop) + Delta H degrees(stem)) and entropic (Delta S degrees = Delta S degrees(loop) + Delta S degrees(stem)) contributions, which permits insightful comparison with predictions from nearest-neighbor thermodynamic models for DNA duplex formation. In particular, the stem thermodynamics is consistent with exothermically favored (Delta H degrees(stem) < 0) and entropically penalized (Delta S degrees(stem) < 0) hydrogen bonding but with additional enthalpic (Delta H degrees(loop) > 0) and entropic (Delta S degrees(loop) > 0) contributions due to loop bending effects consistent with distortion of dA base stacking in the 40-dA linker.