Long-term production of murine hematopoietic cells in vitro is dependent on establishment of a complex microenvironment consisting of a variety of stromal cells and an extensive extracellular matrix which includes collagen, fibronectin, laminin, proteoglycans, and other undefined components adherent to the culture dishes. Cis-4-hydroxyproline (CHP), a relatively specific inhibitor of collagen secretion, was used to examine the role of extracellular collagen deposition in supporting hematopoiesis in long-term C57B1/6J mouse bone marrow cell cultures. Throughout the 10-wk culture period, all culture dishes contained either 0, 10, 25, or 50 micrograms/ml of CHP. All medium and nonadherent cells were removed at weekly intervals and replaced with fresh medium containing the previous concentrations of CHP. Nonadherent cells were assayed weekly for total cells and pluripotent, erythroid, megakaryocytic, and granulocytic-macrophage progenitor cells. Dishes were killed at selected intervals to assess protein and collagen synthesis in the adherent layer. Adherent cell numbers, as judged by microscopic examination and DNA assays, correlated inversely with CHP concentrations used and paralleled degree of collagen synthesis inhibition. The decreased hemopoietic progenitor cell production correlated closely with percent inhibition of collagen synthesis and stromal cellularity. The CHP concentrations tested were not directly toxic to hemopoietic progenitor cells. These studies demonstrate that collagen deposition in the extracellular matrix of murine bone marrow cell cultures is essential to the establishment of a functional stromal microenvironment that is supportive of long-term hematopoiesis.