Many biological systems, especially those based on nucleic acids, are structurally heterogeneous in solution. We demonstrate here the ability to measure multiple distances, of between 2 and 7 nm, from a heterogeneous mixture of double-spin-labeled DNA duplexes. We have constructed a DNA distance ruler based on the attachment of nitroxide spin labels to 2'-amino-modified nucleosides. The distribution of distances between the spin labels was obtained by Tikhonov regularization analysis of the dipolar coupling evolution data measured by using the electron paramagnetic resonance method, pulsed-electron double resonance (PELDOR). Optimization of the conditions and techniques used in the preparation of the samples has allowed us to increase the sensitivity and reduce aggregation artifacts. As a result, we have been able to demonstrate deconvolution of distances from structurally heterogeneous samples and show the limits of the technique by examining data derived from up to five DNA duplexes, in a single mixture, in which the concentration of each species was as low as 5 microM.