To clarify the thermodynamics in the folding phase-transition of single DNA molecules, we studied the effect of temperature on the bimodal distribution of conformation for the ensemble of T4DNA chains (166 kbps) at a fixed concentration of poly(ethylene glycol) using single-chain observation with fluorescence microscopy. From the van't Hoff-type relationship, the entropy change in the unfolding transition is Delta S = + 11.3 k/molecule, and the change in the heat content is Delta H = + 4.77 X 10(-20) J/molecule at T-c = 306 K, where k is the Boltzmann's constant. The positive value of Delta S with the unfolding transition is discussed in relation to the increase in the translational entropy of counterions.