Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection, in particular, the spatial extent of expression in the target tissue and the longevity of expression. Thus, the development of non-invasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression. We now report the design, synthesis and evaluation of a novel in vivo gene transfection reporter molecule 3-O-(beta-D-galactopyranosyl)-6-fluoropyridoxol (GFPOL) using fluorinated vitamin B(6) as the (19)F NMR sensitive aglycone. GFPOL exhibits the following strengths as an in vivo (19)F NMR gene expression reporter: (a) large chemical shift response to enzyme cleavage (Deltadelta=8.00 ppm); (b) minimal toxicity for substrate or aglycone; (c) good water solubility; (d) good blood stability; (e) pH responsiveness of aglycone.