Restriction enzymes produced by bacteria have the unique ability to locate and bind specific stretches of DNA sequence among many millions of bases. The EU-funded ‘Novel approaches to the study of enzymatic diffusion on single DNA molecules’ (SMDNA) project was designed to investigate the mechanism by which enzymes achieve this remarkable feature. A key aspect of the SMDNA proposal involved the sequence-specific fluorescent labelling of DNA using a DNA methyltransferase enzyme. Scientists used the genome of the bacteriophage lambda to label and directly visualise the binding of the structurally well characterised BamHI and BstYI enzymes. Using fluorescent single-molecule imaging, they were able to map the binding of these enzymes as they moved on a single DNA molecule. Through collaboration with a leading DNA enzyme supplier, SMDNA researchers also examined whether heterodimer formation affected the diffusional behaviour of restriction enzymes. Following visualisation at the single molecule level, preliminary analysis showed that enzymes working as heterodimers displayed similar behaviour to other previously studied enzymes. SMDNA results provided important basic knowledge regarding the effect of DNA topology on the diffusion of restriction enzymes and will help enhance understanding of how enzymes interact with DNA in the cell. Furthermore, the developed DNA labelling technology is expected to receive widespread use in the field of molecular biology.