DNA interstrand cross-links (ICLs) are very toxic to dividing cells, because they induce mutations, chromosomal rearrangements and cell death. Inducers of ICLs are important drugs in cancer treatment. We discuss the main properties of several classes of ICL agents and the types of damage they induce. The current insights in ICL repair in bacteria, yeast and mammalian cells are reviewed. An intriguing aspect of ICLs is that a number of multi-step DNA repair pathways including nucleotide excision repair, homologous recombination and post-replication/translesion repair all impinge on their repair. Furthermore, the breast cancer-associated proteins Brca1 and Brca2, the Fanconi anemia-associated FANC proteins, and cell cycle checkpoint proteins are involved in regulating the cellular response to ICLs. We depict several models that describe possible pathways for the repair or replicational bypass of ICLs.