Concentrations of collagenase inhibitor in amniotic fluid correlated with gestational age and with indices of fetal lung maturity such as the lecithin/sphingomyelin ratio and the presence of phosphatidylglycerol. Possible sources of amniotic fluid collagenase inhibitor were sought, and a number of tissues and fluids, both maternal and fetal in origin, were found to contain or synthesize this glycoprotein in significant quantities. The highest concentrations were achieved in cultures of lung fibroblasts implicating the fetal lung as a major source. Although collagenase inhibitor is largely present in a free or available state, its specific role, other than that of a general antiproteinase, has not been determined in amniotic fluid. However, the quantitation of amniotic fluid collagenase inhibitor provides an index of the maturation of the connective tissue of the fetal lung and may reflect the ability of the extracellular matrix to meet neonatal demands.