We report the NMR solution structure of (+)-CPI-indole (CPI, 1,2,8,8a-tetrahydrocyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one), an agent belonging to the CC-1065/duocarmycin family of antitumor compounds. This (+)-CPI-indole structure is covalently bound to d(G(1)ACTAATTGTC(11))-d(G(12)TCAATTAGTC(22)), a synthetic DNA duplex containing a high-affinity binding site. The three-dimensional structure has been determined by several cycles of restrained molecular dynamics calculations with a total of 563 NMR-derived constraints, both in vacuo and by using the generalized Born solvent continuum model. In-depth analysis of the structure of this ligand-DNA complex led to a detailed knowledge of the bound state conformation of the CPI-indole, the most simplified agent related to CC-1065 and duocarmycins, the parent members of a family of extremely potent antitumor compounds. Comparison of the CPI-indole bound conformation with those previously found for (+)-duocarmycin SA (DSA), its unnatural enantiomer (-)-DSA, and the demethoxylated analogue (+)-DSI in their DNA complexes provided additional evidence of the tight correlation between the catalytic effect exerted by DNA on the alkylation reaction and the extent of angular twist between the two planar heteroaromatic subunits of these agents. Additionally, comparison of the structural features of the DNA-bound state of a "naked" ligand, such as CPI-indole, with those of various other duocarmycin agents provided useful information for the interpretation of the observed effects on chemical reactivity of the different substitution patterns at the hemispheres of these types of complex.