Dirhodium tetraacetate is an antitumor active compound that is known to inhibit cellular DNA synthesis, but relatively little is known about its interactions with nucleic acids. A combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and enzymatic digestion experiments has established that the dirhodium bis-acetate unit forms an adduct with AA and GG sites in short DNA strands. The MALDI MS detection of the dirhodium/DNA adduct was aided by the addition of spermine, but no special sample treatment was required to obtain high quality mass spectra of the corresponding cisplatin-modified DNA strands.