Description: Cisplatin is a widely used anticancer drug that acts by binding DNA and causing the formation of intrastrand and interstrand (ICL) crosslinks, but the precise downstream effects of the latter damage are not well understood. In this study, we investigated the influence of cisplatin ICLs on synthetic nucleosomes that were platinated in a site-specific manner in vitro and on gene transcription in live mammalian cells. Nucleosome core particles that we constructed contained site-specific cisplatin 5′-d(G*pC)/5′-d(G*pC) ICLs, where the asterisk denotes the platinated nucleoside, to examine the influence of platinum lesions on the dynamic behavior of nucleosomes in solution. A cisplatin ICL, but not a 1,2-d(GpG) crosslink, significantly inhibited ATP-independent histone octamer-DNA sliding. We also used a novel linearization-recircularization strategy described here to synthesize mammalian expression vectors containing site-specific cisplatin ICLs. Plasmid vectors were tested in live mammalian cells to study the transcription inhibition effects of cisplatin ICLs in the context of two different repair backgrounds. Cisplatin ICLs inhibit transcription as effectively as 1,2-d(GpG) crosslinks. We determined that nucleotide excision repair plays a key role in the removal of cisplatin ICLs, acting in a replication-independent fashion. We also found that loss of mismatch repair function dramatically attenuates the transcription inhibition effects by cisplatin ICLs but not 1,2-d(GpG) intrastrand crosslinks. Our results revealed the unique properties of cisplatin ICLs on nucleosome mobility and on transcription, and they defined how these adducts act in a manner completely different from that used for cisplatin 1,2-d(GpG) crosslinks. These new findings provide direct support for a role of ICLs in the pharmacologic activities of cisplatin, despite the lower frequency of their formation. Cancer Res; 73(14); 4451–60. ©2013 AACR.