The structure of a DNA octamer d(TTGGCCAA)2 complexed to chromomycin-A3 and a singledivalent cobalt ion has been solved by using the pseudocontact shifts due to the unpaired electrons on thecobalt. A protocol was developed and critically evaluated for using the pseudocontact shifts in structuredetermination. The pseudocontact shifts were input as experimental restraints in molecular dynamics simulationswith or without NOE constraints. Both the magnitude and orientation of the susceptibility anisotropy tensorrequired for the shift calculations were determined during the simulations by iterative refinement. Thepseudocontact shifts could be used to define the structure to a very high precision and accuracy comparedwith a corresponding NOE-determined structure. Convergence was obtained from different starting structuresand tensors. A structure determination using both NOE's and pseudocontact shifts revealed a general agreementbetween the two data sets. However, some evidence for a discrepancy between NOE's and pseudocontactshifts was observed in the backbone and terminal base pairs of the DNA. Violations in shift or NOE restraintsremaining in the final structures were examined and may be a reflection of motional averaging of the constraintsand evidence for flexibility. This work demonstrates that pseudocontact shifts are a powerful tool for NMRstructure determination.