It has recently been shown that the anti-cancer drug Adriamycin forms drug-DNA adducts which function as 'virtual' interstrand cross-links in cells, and these cross-links are specific for GpC sequences. The objective of this work was to determine whether all GpC sites are equally susceptible to the formation of Adriamycin-DNA adducts in the mitochondrial genome or whether any 'hotspots' exist whereby lesions are formed preferentially at particular GpC-containing sequences. The mitochondrial genome was used as a model system as it provides a series of contiguous genes, all of which lack introns and in which transcription is driven from a single promoter. With the absence of nucleotide excision repair, this provides an excellent system with which to observe Adriamycin-induced DNA damage since such lesions are reflected as an inhibition of mitochondrial protein synthesis. HeLa cells were treated with Adriamycin and the extent to which synthesis of individual mitochondrial-encoded proteins was inhibited was quantitated. Mitochondrial protein synthesis was found to be inhibited in a discontinuous manner, corresponding to regions rich in 5'-GpC sequences. These results therefore indicate that Adriamycin-DNA adducts do not form randomly with GpC sites throughout the mitochondrial genome, but instead appear to form preferentially at regions of high GpC content. This selective inhibition of mitochondrial-encoded proteins demonstrates the potential of this method for the in situ detection of localized regions of binding by DNA-acting drugs.