Treatment of myofibrils isolated from glycerol-extracted rabbit psoas mucles with dialyzed iron in the pH range of 1.5–2.0 produces a marked deposition of stain particles in the I bands. A bands are considerably less reactive, although M line staining is observed. Control preparations treated with a solution of MgCl 2 at the same pH, concentration, and temperature, and for the same period of time as used for the dialyzed iron reagent are unreactive throughout the myofibrils. Methylation for 4 hours at 60°C, blocks dialyzed iron staining. Extraction of rabbit psoas muscles with low ionic strength Tris buffer, pH 8.0, alters thick filament and thin filament fine structure and removes M lines and Z disks if prolonged sufficiently, but does not prevent dialyzed iron staining of material remaining in the I bands. Digestion of myofibrils with trypsin for 10 minutes removes M lines and Z disks and causes thin filament disorganization in the I bands, but does not prevent reaction of dialyzed iron with material remaining in the I bands. It is concluded that dialyzed iron reveals acid mucosubstances associated with I band thin filaments in rabbit skeletal muscle; it seems unlikely that this reagent reacts nonspecifically with proteins of the myofibrils. It is suggested that acid mucosubstances form a three-dimensional network throughout the I bands.