During ischemia the cardiac stress protein, α B-crystallin, was shown by immunoelectron microscopy to translocate to the N₂-line area of myofibrillar I-bands of rat cardiomyocytes where α B-crystallin resisted extraction with 1 m NaSCN and 2 m urea as did titin. Actin became completely extracted under these conditions, indicating association of α B-crystallin with titin the only remaining non-actin cytoskeletal component of I-bands outside Z-disks. Titin, extracted from ischemic pig myocardium, was shown to copurify with α B-crystallin. Further evidence for binding of α B-crystallin to titin was obtained by dot-blot assays in which biotinylated α B-crystallin was demonstrated to bind to the titin-enriched fraction immobilized on nitrocellulose. Binding of α B-crystallin to titin during cardiac ischemia could serve to stabilize titin against denaturation and might provide an endogenous mechanism to delay ischemic damage of this important elastic component of myofibrils.