I. INTRODUCTION Torsionally unconstrained, double-stranded DNA under longitudinal tension undergoes an abrupt elongation or ‘overstretching’ at about 65 pN, attributed either to melting [1–3] or to the emergence of an elongated, hybridized state called S-DNA [4–8]. Recent elegant experiments [9] show directly that stretched, torsionally unconstrained molecules strand-separate or ‘unpeel’ near 65 pN, an observation argued to rule out the existence of S-DNA. Here we argue instead that the S-DNA picture is consistent with the observation of unpeeling during stretching. Specifically, we demonstrate that this picture predicts that at low temperatures, high salt concentrations and when large forces are applied only for short times, S-DNA may be observed, but that under the converse conditions – those probed in Ref. [9] – unpeeling will coexist with or occur in preference to S-DNA. We argue further that dynamic coexistence of S-DNA and unpeeled DNA can rationalize several recent experimental observations. The essence of the S-DNA picture of overstretching [4–8] is the conjecture that there exists an elongated, hybridized phase of DNA distinct from B-DNA. While cartoon sketches of this picture may convey the idea that it predicts overstretching to consist of the complete conversion of the molecule from B- to S-form, thermodynamic and kinetic analysis [6, 10, 11] predicts a more complex scenario of dynamic coexistence between hybridized and unhybridized forms of DNA. The nature of this coexistence is strongly dependent upon conditions of salt and temperature – low salt concentrations and high temperatures favoring unpeeling – and upon stretching dynamics. Here we use the coarse-grained, statistical mechanical model of Refs. [10, 11] to make predictions for the molecular composition of a 40 kbp fragment of nicked λ -DNA under tension, for a range of environmental conditions, assuming the existence of S-DNA. We find that the thermodynamically- favored composition of DNA under tension (summarized in Fig. 1) is very different from the composition that results if the molecule is stretched dynamically (Figs. 3 to 7). This difference results from the very slow emergence of unpeeled DNA [6]. We find that the instantaneous fraction of S-form DNA varies strongly with stretching force, ambient temperature and salt concentration, and observation time. At the 50 mM salt concentration and (presumably, room) temperatures considered in Ref. [9] we find S-DNA to be very short-lived, and likely unobservable in stretching experiments lasting a few seconds or more. At low temperatures and/or high salt concentra- tions, S-DNA can be long-lived. These predictions are in accord with those of Ref. [6], and with recent experimental evidence for the existence of S-DNA [12]. The latter work identifies two DNA overstretching ‘modes’: a fast mode that operates principally at low temperatures, high salt concentrations and in CG-rich sequences, and a slow mode that proliferates under the converse conditions. These observations were argued to be in accord with the predictions of the S-DNA picture advanced in Ref. [6], with the fast mode reflecting the emergence of S-DNA and the slow mode signaling unpeeling. 2