Monoclonal antibody S5, which is specific for CD44, facilitates engraftment of major histocompatibility complex (MHC)-mismatched canine bone marrow (BM) when infused into recipient animals before total body irradiation (TBI) and marrow infusion. The precise mechanism by which S5 facilitates engraftment is not known. Previously published data in a murine long-term bone marrow culture (LTBMC) model with other anti-CD44 monoclonal antibodies (mAbs) demonstrated an abrogation of myelo- and lymphopoiesis in LTBMC. To address this issue in an in vitro setting, the effect of S5 on canine myelopoiesis in LTBMC was investigated. The data indicate that treatment of LTBMC with S5 causes an absolute increase in the number of progenitor cells as measured by a colony-forming unit-granulocyte/macrophage (CFU-GM) assay compared to cultures treated with control mAb (p < 0.0001). This effect was not observed with three other anti-CD44 mAbs used (Hermes-1, S3, and IM7). In addition, a concomitant decrease in the production of nonadherent cells was noted (p < 0.0001). These effects were evident in both autologous and allogeneic LTBMC systems. mAb S5 has been shown to enhance natural killer (NK) activity, and more recent data indicate the increased cytoxic effect to be partially mediated through CD18. Thus, a possible role for modulation of the CD18 antigen in LTBMC by S5 was assessed by addition of the anti-CD18 mAb 60.3. While the S5-induced reduction in total nonadherent cell production was not changed by addition of 60.3, the increase in progenitor cells stimulated by S5 was abrogated. These findings suggest a role for anti-CD44 antibody in changes in the marrow microenvironment that may be responsible for facilitation of donor engraftment and, at least in part, CD18 may be involved in this phenomenon. The establishment of this in vitro LTBMC model will enable the mechanism to be further dissected.