The effects of exposure of bone marrow to specific methotrexate (MTX) concentrations were studied by constant infusion of the drug into C57BL mice. The residual marrow nucleated cell count was determined in 168 mice at specific intervals. In vitro culture of colony-forming cells (CFU-C) was also performed in 69 of these mice. Duration of exposure varied from 12 to 72 hr. Plateau plasma MTX concentrations were studied in the range from 10-8 to 10-5 m. The total number of nucleated cells per femur fell to a nadir of 30% of control for all drug concentrations studied. The nadir was reached earliest with the highest drug concentrations. The percentage of CFU-C per 7.5 × 104 nucleated cells plated increased after 48-hr infusions compared to the percentage after 24-hr infusions. This increase was seen at all plasma concentrations studied. The total number of CFU-C per femur at plasma MTX concentrations above 10-6 m decreased in the first 24 hr to 40% of control, but then the number significantly increased to 66% of control between 24 and 48 hr. In contrast, no change was observed in CFU-C per femur between 24 and 48 hr during constant infusion at plasma concentrations below 10-6 m. Wright's-stained smears showed no change in the differential count of marrow specimens at 24 and 48 hr that might account for the increased percentage of CFU-C at 48 hr. The increase in CFU-C per femur during high-dose infusions is probably the result of recruitment of CFU-C. The increased percentage of CFU-C suggests recruitment at the lower concentrations as well, but selective elimination of non-CFU-C cells cannot be excluded. Marrow [6-3H]deoxyuridine incorporation studies in vivo during exposure to 10-7 m MTX showed that the phenomenon of recruitment observed in vitro was initiated during the absence of DNA synthesis in vivo.