We have developed an in vitro differentiation assay allowing the study of thymopoiesis from rat bone marrow cells. In this assay, Wistar rat bone marrow cells repopulated fetal Swiss mouse thymic lobes depleted in endogeneous lymphoid cells by deoxyguanosine treatment. Due to the xenogeneic situation, repopulating rat cells from any hemopoietic lineage could be easily recognized by anti-rat monoclonal antibodies such as anti-Thy-1.1 that did not react with Swiss mouse thymocytes. After 15 days in vitro, 80% of the developing rat cells were Thy-1.1+ lymphoid cells and about 70% of the Thy-1.1+ cells expressed CD5, CD2 and leukosialin. The percentages of cells expressing pre-B cell, B cell and myeloid determinants were less than 20%. The developing thymocytes comprised CD4-CD8- T cell receptor (TcR) alpha/beta-, CD4-CD8+TcR alpha/beta low and CD4+CD8+TcR alpha/beta low cells, indicating that the early stages of rat thymopoiesis occurred within mouse thymic lobes. Limiting dilution assays showed that 50% of positive assays were obtained with 3000 nucleated bone marrow cells, which is in good agreement with recent estimates derived from in vivo reconstitution after intrathymical transfer. Moreover the limiting dilution assays proved to be sensitive enough to evidence a tenfold enrichment of pre-T cell activity in the low-density fraction of rat bone marrow. This xenogeneic system might greatly facilitate studies on prethymic and intrathymic stages of rat T cell development and permit new in vitro approaches of the colonizing bone marrow T cell precursor properties.