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Background and Purpose: Perfusion imaging is used for patient selection in clinical practice and trials. Postprocessing and definitions of tissue viability are nevertheless not standardized. We compared the lesion volumes generated with two well-recognized perfusion tissue definitions in a single-center phase 2 thrombolysis study. Methods: We analyzed perfusion imaging data from the Alteplase-Tenecteplase Trial Evaluation for Stroke Thrombolysis (ATTEST) study using two popular tissue viability thresholds (ischemic core definition: (1) cerebral blood volume 2 seconds; penumbra definitions: (1) mean transit time > 145% of contralesional hemisphere or (2) relative delay time < 2 seconds). We compared volumes of core and penumbra, mismatch ratio, percentage, and volume of penumbra salvaged at 24 hours. Results: We included 73 (tenecteplase = 36, alteplase = 37) patients who had analyzable perfusion lesions at baseline. Significant differences were found in core volumes using the two thresholds (33 ± 37 mL vs. 26 ± 32 mL, P < .001), as was mismatch ratio (2.5 ± .9 vs. 4.2 ± 3.7, P < 0.001). The volume of penumbra salvaged at 24 hours (30 ± 19 mL vs. 35 ± 26 mL, P = .043) differed significantly, although the percentages of penumbra salvaged did not (P= .2). No difference was found between the two thrombolytic agents in the percentages of penumbra salvaged using either threshold. Conclusion: Two commonly used tissue definitions generated significantly different lesion volumes and mismatch ratios. Threshold selection may have significant impact on patient selection for trials or reperfusion therapies.