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Abstract Background Suboptimal temporal sampling of time-activity curves (TAC) from dynamic 18F-fluoromethylcholine (FCH) PET images may introduce bias in quantification of FCH uptake in prostate cancer assessment. We sought to define an optimal temporal sampling protocol for dynamic FCH PET imaging. Seven different time samplings were tested: 5 × 60″, 10 × 30″, 15 × 15″–1 × 75″, 6 × 10″–8 × 30″, 12 × 5″–8 × 30″; 10 × 5″–4 × 10″–3 × 20″–5 × 30″, and 8 × 3″–8 × 12″–6 × 30″. First, the irreversible and reversible one-tissue compartment model with blood volume parameter (VB) (respectively, 1T1K+VB and 1T2k+VB, with K1 = transfer coefficient from the arterial blood to the tissue compartment and k2 = transfer coefficient from the tissue compartment to the arterial blood) were compared for 37 lesions from 32 patients who underwent FCH PET imaging for initial or recurrence assessment of prostate cancer, and the model was selected using the Akaike information criterion. To determine the optimal time sampling, K1 values extracted from 1000 noisy-simulated TAC using Monte Carlo method from the seven different time samplings were compared to a target K1 value which is the average of the K1 values extracted from the 37 lesions using an imaging-derived input function for each patient. K1 values extracted with the optimal time sampling for each tumoral lesion were compared to K1 values extracted from each of the other time samplings for the 37 lesions. Results The 1T2k + VB model was selected. The target K1 value as the objective was 0.506 mL/ccm/min (range 0.216–1.246). Results showed a significant difference between K1 values from the simulated TAC with the seven different time samplings analyzed. The closest K1 value from the simulated TAC to the target K1 value was obtained by the 12 × 5″–8 × 30″ time sampling. Concerning the clinical validation, K1 values extracted from the optimal time sampling (12 × 5″–8 × 30″) were significantly different with K1 values extracted from the other time samplings, except for the comparison with K1 values extracted from the 10 × 5″–4 × 10″–3 × 20″–5 × 30″ time sampling. Conclusions A two-phase framing of dynamic PET reconstruction with frame durations of 5 s (blood phase) and 30 s (tissue phase) could be used to sample the TAC for uptake quantification in prostate cancer assessment.