This work describes an optical technique for characterization of the early stages of hematopoietic stem cell (HSC) engraftment under physiological conditions and in real time. Bone marrow cells (BMCs) labeled with PKH membrane linkers were injected into conditioned recipients (B10-->B10.BR mice) preoperated for placement of optical windows over femoral epiphyses. Labeled cells were tracked in vivo by fluorescence microscopy. Cellular adhesion to the BM stroma was tested with laser tweezers, and viability was assayed by the propidium iodide (PI) exclusion test, as determined from energy-transfer measurements of the pair PKH67-PI in freshly excised femurs in situ. At optimal concentrations for in vivo tracking, 1-4 micro M PKH dyes neither impaired the viability of BMCs nor the capacity of allogeneic HSCs to reconstitute hematopoiesis in myeloablated recipients. The optical window allowed in vivo visualization of 23%-26% of the PKH-labeled BMCs in the femur. The homing efficiencies at 16 hours posttransplantation were quantified as 1.77% +/- 0.15% and 0.21% +/- 0.02% for syngeneic and allogeneic BMCs, respectively. In femurs excised 16 hours after transplantation, 70% +/- 9% of the cells were adherent to the BM stroma, and two-thirds of the cells were PI negative (viable). In vivo tracking and in situ assessment of labeled HSCs in recipient BM provide important quantitative and qualitative insights into the early stages of engraftment. Correlation of early events and the efficiency of durable engraftment serve as the basis for a systematic approach toward optimization of the conditions for transplantation.