RTL1000 is a partial human MHC molecule coupled to a human myelin peptide. We previously demonstrated that RTL1000 was protective against experimental ischemic stroke in HLA-DR2 transgenic (DR2-Tg) mice. Since thrombolysis with recombinant tissue plasminogen activator (t-PA) is a standard therapy for stroke, we determined if RTL1000 efficacy is altered when combined with t-PA in experimental stroke. Male DR2-Tg mice underwent 60 min of intraluminal middle cerebral artery occlusion (MCAO). t-PA or vehicle was infused intravenously followed by either a single or four daily subcutaneous injections of RTL1000 or vehicle. Infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride staining at 24 or 96 h of reperfusion. Our data showed that t-PA alone reduced infarct size when measured at 24 h but not at 96 h after MCAO. RTL1000 alone reduced infarct size both at 24 and 96 h after MCAO. Combining RTL1000 with t-PA did not alter its ability to reduce infarct size at either 24 or 96 h after MCAO and provides additional protection in t-PA treated mice at 24 h after ischemic stroke. Taken together, RTL1000 treatment alone improves outcome and provides additional protection in t-PA-treated mice in experimental ischemic stroke.