Background and Purpose-Tissue substrates for action of neuroprotective agents may be absent in a significant proportion of strokes. Pathophysiological heterogeneity is a possible contributor to negative neuroprotective trials. Methods-Stroke subtypes and their individual outcomes in neuroprotective trial control populations were used to derive models incorporating accuracy of clinical classification and probability of an ischemic penumbra. With the use of treatment effect sizes from successful trials (predominantly of reperfusion therapies), sample sizes for neuroprotective trials were calculated. The potential influence of altered recruitment strategies was explored. Results-The proportion of informative patients in 2 large neuroprotective trials was probably only 27% to 30%. Optimistically, this proportion may be 50%; pessimistically, it may be only 17%. These figures necessitate a sample size of 3700 to 4500 subjects per group; at best, 1800 to 2200 are needed per group with optimistic assumptions about treatment effect. Strategies to enhance the proportion with tissue substrate for neuroprotection could reduce sample size to 500 per group and simultaneously reduce the total number of patients screened compared with inclusive trials. Conclusions-Population heterogeneity alone may be sufficient to explain negative neuroprotective trials because even in the largest trials to date sample size is inadequate to detect effect size equivalent to those with thrombolysis, and it is possible that they have been severely underpowered. Reliable trials with inclusive entry criteria may be too large to be commercially feasible for novel compounds. Both sample size and total number of patients needing to be screened should be reduced by restricting entry to patients more likely to have a tissue target.