Aims: To investigate the effect of green flourescent protein (GFP) incorporation on bacterial physiology. Methods and Results: Comparisons were made between four different isogenic pairings of non-GFP-containing parents and their GFP-containing transformants with respect to growth rate and antimicrobial susceptibility. For the latter, sensitivities to 12 different antibiotics were measured initially by disc-diffusion assay, and then subsequently by generation of dose-dependent survival curves for I h exposure to different concentrations of tetracycline, ciprofloxacin and cetrimide USP. Whilst no significant difference in growth rate was observed, GFP-containing strains were uniformly and significantly more sensitive to all antimicrobial agents tested, excluding the beta-lactams, than their respective non-GFP-containing counterparts. Conclusions: GFP incorporation has a significant effect on bacterial physiology and can modulate antimicrobial susceptibility. Significance and Impact of the Study: Transformation with GFP can affect the physiology of bacterial cells. This may therefore affect the quality and accuracy of data generated depending on the application for which GFP is used.