After bone marrow transplantation (BMT), the recipient and donor cells must be distinguished from each other to document and characterize successful engraftment. In addition to dot blot and Southern blot analyses, we have performed in situ hybridization in two sex-mismatched cases using a Y-chromosome specific DNA probe (PHY10). In situ hybridization showed that greater than 95% of the peripheral mononuclear cells had clusters of grains indicative of male cell origin in a recipient girl (case 1), and no cells had clusters of grains in another recipient boy (case 2) at the time of engraftment and 3 months after BMT. In situ hybridization using the PHY10 probe appears to facilitate identification of individual cells of male and female origin, and it requires only 20 hr to obtain the results. The technique provides a powerful new method for the documentation of engraftment and the detection of mixed hematopoietic chimerism in peripheral blood and bone marrow cell compartments after BMT.