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An autoradiographic study was made using S(35)-sulfate for the localization, distribution, and variation in the mucopolysaccharide content of the femoral periosteum of rats from birth to old age. The mucopolysaccharides were also studied histochemically, using toluidine blue O, Rinehart and Abu'l-Haj's colloidal iron method, and the periodic acid-Schiff reaction, before and after hyaluronidase treatment. Autoradiograms revealed the uptake of S(35) particularly in the vicinity of the preosseous zone and adjacent osteoblasts. This labelling was highest at the period of rapid bone growth. With increasing age, the S(35) uptake became progressively less. The preosseous zone showed gamma-metachromatic staining at all ages after treatment with toluidine blue. Active osteoblasts were mostly orthochromatic, however, beta-metachromasia was exhibited at a later age. Abundant amounts of intra- and extracellular mucopolysaccharides of both the acid and neutral type were demonstrated in the periosteum. S(35) uptake and gamma-metachromasia show the presence of sulfated mucopolysaccharides, of which chondroitin sulfate predominates in the preosseous zone. Since S(35) uptake is high in active osteoblasts, the inability to demonstrate metachromasia in osteoblasts may indicate either that chondroitin sulfate is liberated as fast as it is being produced, or that it may be present within the cells in a precursor form not detectable by histochemical methods.