The influence of hypophysectomy (hypox), GH, and TSH on the discrete phases of matrix-induced endochondral bone differentiation was investigated. [3H]Thymidine incorporation by proliferating mesenchymal cells on day 3 was inhibited by hypox, but was corrected by GH administration. On day 7, 35SO4 incorporation into cartilage proteoglycans was reduced by hypox, but was restored to values higher than controls by GH. Calcification of cartilage and bone was monitored by alkaline phosphatase activity. 45Ca incorporation into bone mineral, and total calcium. Alkaline phosphatase levels were maximal on day 11 in the controls and declined thereafter; however, the activity of alkaline phosphatase remained elevated in hypox recipients. Hypox reduced and delayed the rate and extent of calcification, as reflected by 45Ca incorporation and total calcium, respectively. The administration of GH and TSH alone and in combination restored 45Ca incorporation to control values in tibial metaphyses but not in the matrix-induced osteogenic plaques on day 10. These findings imply that the hormonal requirements for initiation of de novo mineralization of bone may be different from those required for the maintenance of mineralization that was initiated in early fetal life, as in the case of metaphyses. Hypox resulted in a delayed and reduced bone formation due to 1) inhibition of mesenchymal cell proliferation, 2) decreased and delayed chondrogenesis, 3) delayed and reduced vascular invasion, and 4) impaired bone formation.