The mechanism of physiologic alteration by high (HDR) or low dose rate (LDR) (5 or 120 cGy/min) irradiation of plateau-phase bone marrow stromal cell cultures was investigated using a technique of in vitro bone marrow transplantation. Purified stromal cell cultures from C57BL/6J, C3H/HeJ, or (C57BL/6J X DBA2/J)F1 (B6D2F1) mouse marrow were irradiated to doses of 2.5 to 10 Gy at LDR or 10-100 Gy at HDR and were then engrafted in vitro with nonadherent hematopoietic cells from murine continuous bone marrow cultures. Parameters of engraftment quantitated included: (1) numbers of adherent proliferating hematopoietic cell colonies, "cobblestone islands" (2) cumulative production of nonadherent hematopoietic cells over 8 weeks after engraftment, (3) M-CSF, GM-CSF and multi-CSF (IL-3) dependent hematopoietic progenitor cells forming greater than or equal to 50 cell colonies in semisolid medium, (4) cumulative production of CFUs, and (5) number of adherent stromal cells positive for detectable extracellular laminin or collagen type IV (markers of endothelial cells, reticular adventitial cells, or sinus lining cells). There was a decrease in cobblestone island formation between 5 and 10 Gy and this parameter possibly increased at doses of 50 and 100 Gy. There was no difference between HDR and LDR irradiation to 10 Gy. Irradiation to doses above 10 Gy decreased support of engrafted cells forming CFU-GM and CFU-GEMM. Measures of CFUs after 10 Gy were variable but indicated a possible increase with HDR and no effect of LDR at 1 week and a decrease in both HDR and LDR groups at 3 weeks after engraftment. Thus, LDR and HDR irradiation in vitro alter several specific parameters of marrow stromal cell support for engrafted hematopoietic stem cells.