Cartilage is a very challenging tissue to preserve optimally for morphological observation due to the complexity of its extracellular matrix which consists of collagens, proteoglycans and various proteins. Conventional chemical fixation results in chondrocyte shrinkage and rupture as well as dislocation of the cell membrane from the pericellular matrix. Cell organelles are poorly contrasted and the glycoprotein network within the pericellular matrix collapses to densely stained granules. These morphological changes are generally attributed to the loss of extracellular matrix proteoglycans and the high osmolarity of the fixative. Therefore the morphology of cartilage can be improved by the preservation of proteoglycans by including an appropriate cationic dye into the fixative and adjusting its osmolarity. Many studies have described optimal chemical fixation techniques, however there is great variation in the composition and osmolarity of fixatives used, and the results are conflicting. Our aim was to develop a method for the optimal chemical fixation of chondrocytes, their subcellular structures and the extracellular matrix. We studied the ultrastructure of rat growth cartilage fixed with 2.5% glutaraldehyde in 0.05 M sodium cacodylate supplemented with 0.7% Safranin O (SO; optimal osmolarity) and in Karnowsky’s fixative supplemented with 0.1% or 0.7% SO (high osmolarity). We found optimal preservation of cartilage with Karnowsky’s fixative supplemented with 0.7% SO. The chondrocytes were well expanded and their unruptured cell membrane showed projections into the pericellular matrix, which was occupied by proteoglycans in expanded form. The intracellular organelles, including rough endoplasmic reticulum and Golgi apparatus, were well preserved, and numerous secretory vacuoles containing proteoglycans and procollagen were easily observed. In contrast the ultrastructure of growth cartilage fixed in the two other fixatives resembled the ultrastructure of cartilage fixed in the conventional method. Our study indicates that optimal cartilage ultrastructure depends mainly on the preservation of both proteoglycan and protein components.