Thymocytes were propagated in long-term cultures supported by stromal cells of both bone marrow and thymus origin. Interleukin 2 (IL-2) supplementation augmented the cell yield and allowed detailed phenotype analysis. Within 2-3 months of culture a cell population was selected in which the expression of Thy-1 antigen persisted, CD4 and CD8 antigens gradually declined, and Pgp-1 antigen, found on less than 5% of fresh thymocytes, was strongly increased. This cultured cell population (Thy-1.2 origin) contained no detectable spleen colony-forming units (CFU-S) but efficiently repopulated the thymus of Thy-1.1-irradiated congenic mice, indicating the precursor T-cell nature of the population. Upon removal from the stroma, the T cells exhibited poor cytotoxicity towards syngeneic tumor cells. Further propagation with IL-2 in the absence of stroma resulted in the acquisition of cytotoxic ability. Replacement of the horse serum used in the above experiments with fetal calf serum resulted in accumulation of cells expressing B220 antigen. This experimental model provides the means to maintain lymphocyte precursor cells in long-term culture and to further study their differentiation in the absence of stroma, both in vitro and in vivo.