The effect of hypophysectomy on in vitro incorporation of isotope into hydroxyproline and hexosamine of calvaria and cartilage of experimental animals has been measured. Following hypophysectomy, the specific activity of hydroxyproline and hexosamine of cartilage was reduced, while the specific activity of both was increased in calvaria. Thus, cartilage and membranous bone respond differently to hypophysectomy. When synthesis of hydroxyproline during a 6-hr incubation was measured as /xmoles synthesized per 100 mg bone, it was found to be essentially the same in bone from hypophysectomized and control animals. It is suggested that the membranous bone of the calvarium grows primarily by cell division; consequently, a measured quantity of bone from either hypophysectomized or control animals could contain an equal number of cells and synthesize an equal quantity of hydroxyproline. The specific activity of matrix hydroxyproline of bone from hypophysectomized animals was probably increased because of a reduced intracellular pool of precusor. The response to PTE was measured in control and hypophysectomized animals. The initial inhibitory phase of hydroxyproline synthesis measured in in vitro incubation was present in both groups; however, the subsequent rebound increase was attenuated in bone from the hypophysectomized animals. Also, the increase in specific activity of hexosamine following PTE administration to controls was attenuated in the absence of the pituitary. (Endocrinology76: 198, 1965)