4’‐6‐Diamidino‐2‐phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4’‐6‐Diamidino‐2‐phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4’‐6‐Diamidino‐2‐phenylindole will not be observed. In this paper, we show that following observation of 4’‐6‐Diamidino‐2‐phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4’‐6‐Diamidino‐2‐phenylindole together with widely used green fluorophores in double labelling experiments.