The cytotoxic and mutagenic properties of the anticancer drug cis-diammine-dichloroplatinum(II) (cisplatin) are mediated by bifunctional adducts between purines. Experiments performed in this study employed a new repetitive thermal-cycling technique to detect cisplatin adduct formation following exposure of cells in culture (in vivo) or following treatment of purified DNA (in vitro exposure). The initial goal of this study was to determine if cisplatin-DNA adduct formation could be measured accurately using phosphor-imaging over a broad concentration range. If this proved possible, it would then be feasible to determine if adduct formation differed within chromatin compared with purified DNA. There were no significant differences in the cisplatin-DNA adduct pattern induced in closed circular or linear double-stranded plasmids in vitro, suggesting that this type of tertiary structural change does not affect the formation of adduct sites. Sequence-specific DNA adduct formation within a human repetitive DNA target sequence, alphoid DNA, following cisplatin treatment of prostate cancer cells in culture (in vivo) and treatment of purified DNA in vitro revealed consistent increases in adduct formation over a broad concentration range, validating the experimental technique. Comparing preferences for cisplatin adduct site formation under these different conditions of exposure demonstrated statistically significant differences. Similar differences were detected for cisplatin repair-deficient Xeroderma pigmentosum cells treated in cell culture, indicating that in vivo/in vitro preferences for adduct site formation are not the result of DNA repair in vivo.