A human/sheep xenograft model was used to evaluate whether long-term engrafting haematopoietic stem cells (HSC) are susceptible to human cytomegalovirus (HCMV) infection. CD34+ Lin- HSC were isolated by fluorescence-activated cell sorting (FACS) from the bone marrow (BM) of HCMV-positive and HCMV-negative normal donors. Cells from the latter group were infected in vitro with HCMV. HCMV DNA was detected in both cell populations by nested-polymerase chain reaction (PCR) and fluorescence in situ hybridization. Cells were transplanted into separate groups of fetal sheep at concentrations of 1.3-5.0 x 105 cells per fetus. Multilineage human haematopoietic cell engraftment, including CD34+ cells, was detected in the BM and peripheral blood of recipients up to 16 months post-transplant as assessed by FACS analysis and PCR for HLA-DQalpha. Levels of engraftment varied (1.2-24.3%) but no sheep exhibited HCMV-positive cells. To ensure that our inability to detect HCMV-positive cells was not due to immune-elimination of HCMV-infected cells, 3.8-10 x 105 HCMV-positive uncharacterized BM stromal cells were transplanted into fetal sheep. At 5 weeks post-transplant several organs were HLA-DQalpha- and HCMV-positive, confirming that HCMV was detectable. These results provide evidence that the long-term engrafting HSC is not a primary target of HCMV and suggest that HCMV infection of human haematopoietic cells is exercised at the level of committed progenitors.