Human marrow was obtained as material discarded during total hip replacement and was established in culture with phenol red-free alpha-MEM with 10% fetal bovine serum (FBS) and antibiotics. Insulin-like growth factor I (IGF-I) and its binding proteins were secreted by human marrow cells, in amounts that increased with time in culture. Western ligand blotting showed that insulin-like growth factor binding protein-3 (IGFBP-3) accounted for the majority (approximately 75%) of the secreted binding proteins. Evidence for marrow secretion of BP-3 protease was found by electrophoretic analysis of mixtures of radiolabeled IGFBP-3 and marrow-conditioned media. The amount of constitutive secretion of IGFBP-3 increased with age of the subject (r = 0.97, p = 0.0058). A notable exception was marrow from a postmenopausal women on estrogen replacement therapy (ERT) at the time of surgery; her marrow secreted 89.3 ng/mL after 14 d in vitro, only 38% of the IGFBP-3 that was secreted by cultures from two age-matched peers (208.8 and 285.2 ng/mL). This in vivo effect of estrogen was matched by an in vitro experiment in which 10(-8) M 17-beta estradiol suppressed IGFBP-3 to 60% of the constitutive level. In all cultures of marrow from postmenopausal women, IL-1 beta suppressed IGFBP-3 secretion to either undetectable levels or levels between 11% and 35% of control. Thus, human bone marrow cultures demonstrate components of the skeletal IGF regulatory system: IGF-I, IGF-binding proteins, and evidence of IGFBP-3 proteolysis. These results provide evidence of regulation by both systemic (age, estrogen status) and cytokine (IL-1 beta) factors.