Objective: We describe the first clinical application of transient hyperoxia (“oxygen challenge”) during T2*-weighted magnetic resonance imaging (MRI), to detect differences in vascular deoxyhemoglobin between tissue compartments following stroke. Methods: Subjects with acute ischemic stroke were scanned with T2*-weighted MRI and oxygen challenge. For regions defined as infarct core (diffusion-weighted imaging lesion) and presumed penumbra (perfusion-diffusion mismatch [threshold = Tmax ≥4 seconds], or regions exhibiting diffusion lesion expansion at day 3), T2*-weighted signal intensity–time curves corresponding to the duration of oxygen challenge were generated. From these, the area under the curve, gradient of incline of the signal increase, time to maximum signal, and percentage signal change after oxygen challenge were measured. Results: We identified 25 subjects with stroke lesions >1ml. Eighteen subjects with good quality T2*-weighted signal intensity–time curves in the contralateral hemisphere were analyzed. Curves from the diffusion lesion had a smaller area under the curve, percentage signal change, and gradient of incline, and longer time to maximum signal (p < 0.05, n = 17) compared to normal tissue, which consistently showed signal increase during oxygen challenge. Curves in the presumed penumbral regions (n = 8) showed varied morphology, but at hyperacute time points (<8 hours) showed a tendency to greater percentage signal change. Interpretation: Differences in T2*-weighted signal intensity–time curves during oxygen challenge in brain regions with different pathophysiological states after stroke are likely to reflect differences in deoxyhemoglobin concentration, and therefore differences in metabolic activity. Despite its underlying complexities, this technique offers a possible novel mode of metabolic imaging in acute stroke.