Multiprobe equilibrium unfolding experiments in the downhill regime (i.e., maximal barrier < 3RT) can resolve the folding process with atomic resolution [Muñoz (2002) Int. J. Quantum Chem. 90, 1522-1528]. Such information is extracted from hundreds of heterogeneous atomic equilibrium unfolding curves, which are characterized according to their denaturation midpoint (e.g., T m for thermal denaturation). Using statistical methods, we analyze Tm accuracy when determined from the extremum of the derivative of the unfolding curve and from two-state fits under different sets of simulated experimental conditions. We develop simple procedures to discriminate between real unfolding heterogeneity at the atomic level and experimental uncertainty in the single Tm of conventional two-state folding. We apply these procedures to the recently published multiprobe NMR experiments of BBL [Sadqi et al. (2006) Nature 442, 317-321] and conclude that for the 122 single transition atomic unfolding curves reported for this protein the mean Tm accuracy is better than 1.8 K for both methods, compared to the 60 K spread in Tm determined experimentally. Importantly, we also find that when the pre- or posttransition baseline is incomplete, the two-state fits systematically drift the estimated Tm value toward the center of the experimental range. Therefore, the reported 60 K Tm spread in BBL is in fact a lower limit. The derivative method is significantly less sensitive to this problem and thus is a better choice for multiprobe experiments with a broad Tm distribution. The results we obtain in this work lay the foundations for the quantitative analysis of future multiprobe unfolding experiments in fast-folding proteins. © 2008 American Chemical Society.