When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have investigated the source of this transition by altering helix stability with solution pH. As solution pH was increased from pH 6.0 to pH 10.6 in 250 mM NaCl, the overstretching transition force decreased from 67.0 ± 0.8 pN to 56.2 ± 0.8 pN, whereas the transition width remained nearly constant. As the pH was lowered from pH 6.0 to pH 3.1, the overstretching force decreased from 67.0 ± 0.8 pN to 47.0 ± 1.0 pN, but the transition width increased from 3.0 ± 0.6 pN to 16.0 ± 3 pN. These results quantitatively agree with a model that asserts that DNA strand dissociation, or melting, occurs during the overstretching transition.