No / The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of DNA minor groove binding agents that react covalently with guanine bases, preferably at Pu-G-Pu sites. A series of three fluorescent PBD¿coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA binding affinity, cellular localization and cytotoxicity. The results show that the linker structure plays a critical role for all three parameters. Graphical abstract A series of three fluorescent PBD¿coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA-binding affinity, cellular localization and cytotoxicity.