Sincalide-stimulated cholescintigraphy is performed to quantify gallbladder contraction and emptying. However, different infusion methods are used for this study. Our purpose was to determine the infusion method with the least variability (smallest coefficient of variation [CV]) for calculation of the gallbladder ejection fraction (GBEF) in healthy subjects and to establish normal values. METHODS Sixty healthy volunteers at 4 medical centers were injected intravenously with (99m)Tc-mebrofenin. After gallbladder visualization had been confirmed at 60 min, 0.02 microg of sincalide per kilogram was administered using 3 different infusion durations, 15, 30, and 60 min, each performed on separate days. The CV, mean, SD, first to 99th percentile, and fifth to 95th percentile were calculated. GBEF normal values were determined for the different infusion durations. RESULTS The CV was smallest for the 60-min infusion at 60 min (19%; 95% confidence interval [CI], 16%-23%), compared with the 30-min infusion at 30 min (35%; 95% CI, 29.2%-42.1%) and the 15-min infusion at 15 min (52%; 95% CI, 44%-63%). These were all significantly different (P < 0.0007). For the 60-min infusion at 60 min, the lower limit of normal for the GBEF was 38% defined at the 1% CI. CONCLUSIONS The GBEF at 60 min has the lowest CV in healthy subjects, compared with shorter infusions of 15 or 30 min. This multicenter trial establishes a GBEF lower limit of normal of 38% (first percentile) for a 60-min infusion of 0.02 microg of sincalide per kilogram, quantified at 60 min. Using this infusion method minimizes the variability in measured GBEFs. This sincalide infusion method should become the standard for routine clinical use.