Native fluorescence spectroscopy (NFS), primarily from tryptophan (trp), was used for in situ investigation of the virus-receptor attachment process in phi6, a lipid-containing bacteriophage from the Cystoviridae family. NFS allowed us to monitor the viral attachment directly to its receptor, which was isolated from the pseudomonad host. Immediately upon mixing, an increase in tryptophan emission intensity was observed followed by a subsequent decrease in emission intensity. The initial increase in emission intensity reflects changes in trp quantum efficiency as the phi6 surface proteins change their conformation as a result of virus attachment to the pilus. The cystovirus spike protein P3 is responsible for receptor recognition and the fluorescence changes observed are likely to be the consequence of its conformational transition at this initial infection stage, providing a kinetic view of this process. The subsequent decrease in trp emission intensity could be due to changes in viral proteins as a result of disassembly of the pili. The technique may have important applications for the dynamic monitoring of additional stages of the virus replication cycle such as assembly, interaction with nucleic acids and maturation. This work expands on a previous demonstration that fluorescence offered a novel tool to detect virus particle interaction with its host cell.