Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding -galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a ¹⁹F NMR approach to reveal lacZ gene expression by assessing -galactosidase (-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl -D-galactopyranoside (OFPNPG) is readily hydrolyzed by -gal with a corresponding decrease in the ¹⁹F-NMR signal from OFPNPG and the appearance of a new signal shifted 4-6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using ¹⁹F NMR spectroscopy and ¹⁹F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach.--Liu, L., Kodibagkar, V. D., Yu, J-X., Mason, R. P. ¹⁹F-NMR detection of lacZ gene expression via the enzymic hydrolysis of 2-fluoro-4-nitrophenyl -D-galactopyranoside in vivo in PC3 prostate tumor.