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Introduction Noninvasive mapping of enzyme activity with MRI holds a great potential for disease diagnosis and evaluation of cell therapies. Dual-modality approaches are gaining popularity as they deliver complementary information allowing more unambiguous data interpretation on enzyme activity in vivo. Here, we report on such a dual 1H/19F MRI probe (Fig1) responsive to β-gal encoded by LacZ reporter gene [1]. Probe activation leads to "turning on" of the 19F signal (via alterations in 19F T1,T2) and changed ability of the Gd3+ complex to modulate the 1H MR signal intensity of water. Methods Gd-DOMF-Gal was obtained by attachment of galactose and MRI reporters to a self-immolative linker. 19F NMR spectra were recorded on a 300MHz NMR spectrometer (~37°C). The 19F-MR images were acquired on a 7T MRI system with FLASH and true FISP using a dual 1H/19F surface coil (~25°C). T1 maps were obtained by voxel-wise fitting of the 1H signal (IR-FLASH). Relaxivity r1 values were determined at 3T and 7T (~25°C). Results Gd-DOMF-Gal was successfully synthesized. Its 19F T1,T2 values were in the range of 3~6 ms, but could not be estimated precisely due to the broadening and low signal intensity of the 19F NMR peak as an effect of the intramolecular relaxation enhancement by Gd3+. Upon activation, the 19F resonance became sharp and intense with 19F T1 and T2 values of 245.4±2.9 and 182.3±3.5 ms measured for the product. In the 19F MR images no signal was detected for Gd-DOMF-Gal, while a 19F signal of increasing intensity was observed after conversion (Fig2). The corresponding proton T1 maps displayed a significant elongation of T1 in the presence of β-gal. The relative r1 changes (Δr1) upon activation were found to be ~27 in PBS at 7T (6.66±0.62 vs 5.10±0.49mM-1s-1) and 3T (9.55±0.05 vs 7.00±0.10 mM-1s-1), whereas a Δr1 of 46 was observed in HEPES. We also compared true FISP with 19F FLASH sequence in terms of sensitivity and specificity for imaging of probe activation. While an SNR of 14 was determined in true FISP images, compared to 10 in FLASH images, the use of FLASH is still preferable for our approach as it allows unambiguous distinction between substrate and product. Conclusions We have developed a “smart” probe, which activation by β-gal can be measured by 1H/19F MRI. With this molecular design we provide a potential tool for monitoring not only the activity of β-gal, but also other enzymes by means of 1H and 19F MRI.