Rh(phen)2phi3+(phen = 1,10-phenanthroline; phi = 9,10-phenanthrenequinone diimine) avidly binds to DNA via intercalation from the major groove and upon photoactivation produces strand scission with single base 5' asymmetry. Enantiomers of Rh(phen)2phi3+, which lack hydrogen-bonding substituents in ancillary positions, distinguish a DNA site through shape-selection; site recognition depends upon the local variations in structure at a binding site. Here, we examine the application of DELTA-Rh(phen)2phi3+ as a sequence-dependent structural probe and, in particular, as a probe of DNA propeller twisting in solution, by comparing directly cleavage results using DELTA- and LAMBDA-Rh(phen)2phi3+ on crystallographically characterized oligonucleotides with several sequence-dependent crystallographic parameters. The three oligonucleotides examined in this study are the Dickerson-Drew dodecamer, 5'-CGCGAATTCGCG-3', the Nar I dodecamer, 5'-ACCGGCGCCACA-3', and the CG decamer, 5'-CCAACGTTGG-3', all of which have been crystallized in the B-form. Enantioselective cleavage and reaction favored by the DELTA-isomer is found to be governed locally by the opening of the site in the major groove. A correlation is demonstrated between cleavage by DELTA-Rh(phen)2phi3+ and the opening in the major groove that results from the change in propeller twist (differential propeller twist) at a base step. When the major groove is closed as a result of a change in propeller twist, there is little cleavage evident by either enantiomer; at sites which are indicated crystallographically to be open in the major groove, a direct correlation is observed between enantioselective cleavage and the degree of opening. A trend of higher enantioselectivity at sites possessing higher twist angles is also observed. The roll angle of a site in isolation, the rise per base step, and the groove width show no correlation with cleavage. The distribution of reaction products at a site, as a result of partitioning along oxygen-dependent or oxygen-independent pathways, also provides a sensitive measure of the shape-complementarity between metal complex and binding site. These results provide evidence for site-recognition by the metal complex which correlates directly with DNA propeller twisting in oligonucleotide crystals. Importantly, these results indicate that Rh(phen)2phi3+ may be uniquely applied as a chemical probe for sequence-dependent changes in the propeller twisting of DNA.