Radiolabeled prostate-specific membrane antigen (PSMA) PET has demonstrated promising results for prostate cancer (PCa) imaging. Quantification of PSMA radiotracer uptake is desired as it enables reliable interpretation of PET images, use of PSMA uptake as an imaging biomarker for tumor characterization, and evaluation of treatment effects. The aim of this study was to perform a full pharmacokinetic analysis of 2-(3-(1-carboxy-5-[(6- 18F-fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentanedioic acid ( 18F-DCFPyL), a second-generation 18F-labeled PSMA ligand. On the basis of the pharmacokinetic analysis (reference method), simplified methods for quantification of 18F-DCFPyL uptake were validated. Methods: Eight patients with metastasized PCa were included. Dynamic PET acquisitions were performed at 0-60 and 90-120 min after injection of a median dose of 313 MBq of 18F-DCFPyL (range, 292-314 MBq). Continuous and manual arterial blood sampling provided calibrated plasma tracer input functions. Time-activity curves were derived for each PCa metastasis, and 18F-DCFPyL kinetics were described using standard plasma input tissue-compartment models. Simplified methods for quantification of 18F-DCFPyL uptake (SUVs; tumor-to-blood ratios [TBRs]) were correlated with kinetic parameter estimates obtained from full pharmacokinetic analysis. Results: In total, 46 metastases were evaluated. A reversible 2-tissue-compartment model was preferred for 18F-DCFPyL kinetics in 59% of the metastases. The observed k 4 was small, however, resulting in nearly irreversible kinetics during the course of the PET study. Hence, k 4 was fixated (0.015) and net influx rate, K i, was preferred as the reference kinetic parameter. Whole-blood TBR provided an excellent correlation with K i from full kinetic analysis (R 2 = 0.97). This TBR could be simplified further by replacing the blood samples with an image-based, single measurement of blood activity in the ascending aorta (image-based TBR, R 2 = 0.96). SUV correlated poorly with K i (R 2 = 0.47 and R 2 = 0.60 for SUV normalized to body weight and lean body mass, respectively), most likely because of deviant blood activity concentrations (i.e., tumor tracer input) in patients with higher tumor volumes. Conclusion: 18F-DCFPyL kinetics in PCa metastases are best described by a reversible 2-tissue-compartment model. Image-based TBRs were validated as a simplified method to quantify 18F-DCFPyL uptake and might be applied to clinical, whole-body PET scans. SUV does not provide reliable quantification of 18F-DCFPyL uptake.