The conversion of l-[³H]arginine to l-[³H]citrulline in the absence of calcium can be used to assay selectively the activity of inducible nitric oxide synthase (NOS) in rat spleen homogenates 6 h after lipopolysaccharide administration. Using similar assay conditions, changes in inducible NOS activity were measured within ischemic brain tissue between 2 h and 7 days following permanent middle cerebral artery (MCA) occlusion in Sprague-Dawley rats and SV-129 mice. Total (constitutive and inducible) NOS activity was measured in the presence of 0.5 mM CaCl₂. Whereas total NOS activity in rat decreased dramatically to 16% and 6% of baseline 6 and 12 h after MCA occlusion, inducible NOS activity remained undetectable before 2 days after occlusion, became maximal at 3 days, and decreased to less than 10% of maximal iNOS activity at 7 days. In the mouse, total NOS activity decreased after MCA occlusion but inducible NOS activity was undetectable from 2 h to 4 days after occlusion. Sustained NO production by inducible NOS activity does not contribute to ischemic injury within 24 h after MCA occlusion, but may contribute to infarct maturation 2–4 days after ischemia in some but not all species.