Chemical exchange saturation transfer (CEST) is a new approach for generating magnetic resonance imaging (MRI) contrast that allows monitoring of protein properties in vivo. In this method, a radiofrequency pulse is used to saturate the magnetization of specific protons on a target molecule, which is then transferred to water protons via chemical exchange and detected using MRI. One advantage of CEST imaging is that the magnetizations of different protons can be specifically saturated at different resonance frequencies. This enables the detection of multiple targets simultaneously in living tissue. We present here a CEST MRI approach for detecting the activity of cytosine deaminase (CDase), an enzyme that catalyzes the deamination of cytosine to uracil. Our findings suggest that metabolism of two substrates of the enzyme, cytosine and 5-fluorocytosine (5FC), can be detected using saturation pulses targeted specifically to protons at +2 ppm and +2.4 ppm (with respect to water), respectively. Indeed, after deamination by recombinant CDase, the CEST contrast disappears. In addition, expression of the enzyme in three different cell lines exhibiting different expression levels of CDase shows good agreement with the CDase activity measured with CEST MRI. Consequently, CDase activity was imaged with high-resolution CEST MRI. These data demonstrate the ability to detect enzyme activity based on proton exchange. Consequently, CEST MRI has the potential to follow the kinetics of multiple enzymes in real time in living tissue.