We have subdivided mouse CD4-CD8-CD3- triple-negative (TN) thymocytes into four subsets based upon expression of CD44 and CD25, including CD44+CD25-, CD44+CD25+, CD44-CD25+ and CD44-CD25-. Characterization of these cells revealed several features distinct to each subset, in particular the expression of high levels of c-kit (the receptor for stem cell factor) by CD44+CD25-TN and CD44+CD25+TN but not by CD44-CD25+TN and CD44-CD25-TN. The CD44+CD25+TN subset also included the IL-7 and stem cell factor-responsive cells, whereas only minimal responsiveness was observed by the CD44- populations. These subsets also showed differential cytokine production potential (CD44+CD25- > CD44+CD25+ > CD44-CD25+ > CD44-CD25-) after stimulation with calcium ionophore, PMA and IL-1. The repopulation potential of these subsets in 2-deoxyguanosine-treated fetal thymic lobes supports the following maturation sequence: CD44+CD25- -->CD44+CD25+ -->CD44-CD25+ -->CD44-CD25-. Furthermore, the sequence of progression from CD44+CD25+ to CD44-CD25+ cells was confirmed by their TCR beta-chain gene configuration. The former population exhibits germ-line TCR beta-chain configuration, whereas the latter subset shows a rearranged pattern.