Purpose A novel caspase-3 substrate-based probe 18 F-CP18 was evaluated as an in vivo positron emission tomography (PET) imaging agent for monitoring apoptosis in tumors. Methods Uptake of 18 F-CP18 in cell assays and tumors was measured. Caspase-3/7 activities in cell lysates and tumor homogenates were determined. Autoradiography,Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cleaved caspase-3 immunostaining were performed on adjacent tumor sections to identify areas of apoptosis. Results The in vitro cell assays showed caspase-3-dependent uptake of 18 F-CP18 in tumor cells when treated with an apoptosis inducer. The in vivo microPET imaging signal of 18 F-CP18 in xenograft tumors correlated with the ex vivo caspase-3/7 activities in these tumors. Furthermore, tumor autoradiographies of 18 F-CP18 in tumor sections matched adjacent sections stained by TUNEL and caspase-3 immunohistochemistry (IHC). Conclusions 18 F-CP18 demonstrated high affinity and selectivity for activated caspase-3 both in vitro and in vivo , and the results support 18 F-CP18 as a promising new PET imaging agent for apoptosis.