Sl/Sl(d) mice have a defective hemopoietic microenvironment. It has been assumed, based upon previous studies, that the primary abnormality in these mice is simply lack of a necessary supportive or inductive material within the hemopoietic stroma. We used in vitro long-term bone marrow cultures to characterize further the nature of the hemopoietic microenvironmental defect in Sl/Sl(d) mice. Sl/Sl(d) mouse bone marrow cells consistently produced <10% of the total hemopoietic cells and multipotent and unipotent hemopoietic progenitor cells produced in cultures of marrow from normal, congenic +/+ mice. If fresh Sl/Sl(d) and +/+ marrow cells were mixed prior to establishing long-term marrow cultures, there was a direct correlation between number of Sl/Sl(d) cells added and degree of inhibition of +/+ hemopoiesis. A pre-established, confluent Sl/Sl(d) adherent stromal layer inhibited hemopoiesis by fresh +/+ marrow cells by nearly 70%, as compared with dishes with irradiated +/+ or no stroma. This inhibitory effect was abrogated by irradiation of the Sl/Sl(d) stroma prior to addition of the fresh +/+ marrow cells. Similarly, unirradiated, but not 9 to 200 Gy irradiated Sl/Sl(d) stroma inhibited proliferation of the factor-dependent FDC-P1 hemopoietic progenitor cell line. Thus, the Sl/Sl(d) hemopoietic microenvironment actively inhibits hemopoiesis in vitro, and this inhibition can be at least partially eliminated by irradiation of the Sl/Sl(d) stroma.