The cationic stain ruthenium hexammine trichloride (RHT) has been utilized as a connective tissue stain primarily due to its ability to stabilize and stain anionically-charged glycosaminoglycans (GAGs). A major drawback of this procedure however is that the increased preservation and visibility of the GAGs obscures other RHT-stained constituents of the “ground substance”. In order to more readily visualize these non-GAG ground substance components ligament tissue was pre-treated with chondroitinase ABC, an enzyme that removes most, if not all of the GAGs present.Segments of the midsubstance of medial collateral ligaments (MCL) of mature New Zealand white rabbits were excised and incubated 1 hat 37°C in chondroitinase ABC (1 U/ml in 0.25M Tris, 0.18M NaCl, 5mM benzamidine HC1, 0. 1. 6-amino-n-caproic acid, pH 8.0) followed by fixation in Na-cacodylate (0.1M, pH 7.4) buffered glutaraldehyde (2.5%) containing RHT (lmg/ml) for 2.5 h. Post-fixation for 1 h in cacodylate-buffered osmium tetroxide (1%) plus RHT (lmg/ml) preceded ethanol dehydration and embedding in Spurr's resin.