The intrinsically fluorescent green fluorescent protein has been used in many laboratories as a cytological marker to monitor protein localisation in live cells. Multiple spectrally modified mutant versions and novel fluorescent proteins from other species have subsequently been reported and used for labelling cells with multiple fluorescent protein fusions. In this work we report the design and use of vectors containing some of these spectral variants of GFP for use in the Gram positive bacterium Bacillus subtilis. These vectors complement those previously described (Lewis and Marston, 1999. Gene 227, 101–109) to provide a large suite of plasmid vectors for use in this and other related Gram positive organisms. Using these vectors we have been able to directly demonstrate the sequential assembly/disassembly of proteins involved in the generation of cellular asymmetry during development.