Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade structural components of the extracellular matrix and participate in pathologies such as cancer and inflammatory disorders. The development of novel contrast agents for optical imaging of MMP activity in vivo is of great interest. The commonly used near-infrared fluorescence-compatible agents are dye-quenched probes that do not emit fluorescence until they interact with MMPs. In contrast, fluorescent synthetic low-molecular-weight MMP inhibitors have not been systematically employed. The aim of this study was to evaluate the performance of our recently developed Cy5.5-labeled MMP inhibitor to image MMP activity in tumors in vivo compared with activatable fluorescent MMP-sensing probes. The dynamic uptake of Cy5.5-AF489 into four different tumor entities was analyzed in xenografted mice by intravenous injection and subsequent fluorescence reflectance imaging. Tumors were characterized in regard to their MMP-2 and −9 mRNA expressions (qRT-PCR analysis), proteins (immunohistochemistry) and gelatinase/collagenase activities (in situ zymography). Cy5.5-AF489 was compared with MMPSenseTM 680 and MMPSenseTM 750 FAST, two commercially available MMP-activatable probes. Cy5.5-AF489 showed a specific tumor uptake, which was blocked by pre-injection of the unlabeled MMPI, and discriminated between tumors with high or low MMP-2/-9 expressions. Our optical probe facilitated faster visualization of MMP-active tumors accompanied by excellent tumor-to-background ratios when compared with activatable probes. The MMP inhibitor Cy5.5-AF489 permits fast in vivo imaging of MMP expression/activity in tumors. Given its small molecular weight and non-peptidic structure, translational imaging from a preclinical application to a diagnostic tool for MMP-related diseases seems feasible. Copyright © 2012 John Wiley & Sons, Ltd.