Minor thymus subpopulations representing possible intermediates in thymic positive selection were isolated by cell sorting from bcl-2 transgenic mice, and cultured 1 to 4 days in simple medium to assess their ability to spontaneously develop the surface phenotype of mature T cells. Recovery of cells was in the 60 to 80% range, and no cell proliferation occurred. Only cells originally expressing high, near mature T cell levels of CD3 developed further in culture by down-regulation of CD4 or CD8. The main mature cell product was CD4-8+, regardless of whether the starting phenotype of the CD3high intermediates was CD4+8+, CD4int8+, or CD4+8int; only an intermediate subpopulation expressing the highest levels of CD4 (CD4high8int) produced a dominance of CD4+8- mature progeny. Partial down-regulation of CD8 was therefore not a good indicator of CD4+ T lineage commitment. These and previous results indicate that maturation to the CD8+ T lineage involves a rapid up-regulation of the TCR-CD3 complex, but a relatively slow down-regulation of CD4; it may also involve a partial, transient reduction in surface CD8. In contrast, maturation to the CD4+ T lineage involves a relatively rapid down-regulation of CD8, with maintenance of high levels of CD4. There appears to be a marked asymmetry in the developmental steps leading from CD4+8+ thymocytes to the CD8+ or to the CD4+ T cell lineage.