Several murine lymphoid cell lines have been tested for specific capacity to localize in thymus. These are all continuous, cloned Radiation leukemia virus-induced cell lines which have a common phenotype resembling lymphoid stem cells or immature T cells. Since each of these cell lines has a cloning efficiency approaching 100%, the number of cells which enters thymus during a 3 h homing assay has been estimated by limit dilution cloning analysis taking into account extra-binomial variation caused by individual mice. Only two out of seven of these cell lines have been found to have this specific property. These two cell lines, 16C1 and 5C2B, have been characterized as immature lymphoid cells, bearing no rearrangement at the TCR gamma and beta loci, and having the phenotype of CD3+CD4-CD8-, immature T cells. A maximum number of 2000 16C1 cells and 2500 5C2B cells can enter thymus during a 3 h homing assay, suggesting a limited number of sites in thymus to which these cells can bind. The capacity of 16C1 to enter thymus in low frequency has been found to be a stable property and was not increased by repetitive passage through mouse thymus. Using this assay, we have also been able to confirm that entry of 16C1 cells into thymus can be inhibited by antibody specific for the Ly24 (Pgp-1) molecule.