UNLABELLED: Ventilation-perfusion (V/Q) SPECT has been reported to improve the diagnostic performance of V/Q imaging for the diagnosis of pulmonary embolism (PE). However, only sparse data based on an objective reference test are available, and the criteria used for interpretation have varied widely. Therefore, the aim of our study was to assess the performance of V/Q SPECT using various criteria for interpretation, in comparison with a validated independent diagnostic strategy. METHODS: The SPECT study included patients for whom V/Q SPECT data were compared with the results of an independent and validated diagnostic algorithm for PE. V/Q SPECT scans were performed after intravenous injection of (99m)Tc-macroaggregated albumin and simultaneous ventilation with (81m)Kr gas. Interpretation was performed independently by 2 nuclear medicine physicians who were not aware of the clinical history, diagnostic strategy conclusion, or patient's outcome. Sensitivity, specificity, and likelihood ratios were evaluated for various combinations of mismatched defect numbers and sizes (segmental or subsegmental). Generation of receiver-operating-characteristic curves was based on the number of mismatch defects and the number of subsegmental mismatch defects or equivalent. RESULTS: Of the 249 patients who were analyzed, the diagnosis of PE was confirmed in 49 and ruled out in 200 according to the previously validated independent strategy. Of all the tested criteria, the best performance was achieved using a diagnostic cutoff of at least 1 segmental or 2 subsegmental mismatches, with sensitivity and specificity of 0.92 (95% confidence interval, 0.84-1) and 0.91 (95% confidence interval, 0.87-0.95), respectively. With a negative V/Q SPECT result, the posttest probability of PE was 0.010, 0.037, and 0.119 for a low, intermediate, and high clinical probability. With a positive V/Q SPECT result, the posttest probability of PE was 0.531, 0.814, and 0.939 for a low, intermediate, and high probability. CONCLUSION: For V/Q SPECT interpretation, a diagnostic cutoff of 1 segmental or 2 subsegmental mismatches seems best for confirming or excluding acute PE.