We have previously shown that the occurrence of CD45RA+ adult mouse thymocytes is strain-dependent, e.g. constituting approximately 0.6% in C57BL/Icrf and approximately 2.5% in BALB/c (Huby, R. and Goff, L., 1992. Eur. J. Immunol. 22:1659). Here we show that irrespective of strain, the thymus contains approximately 0.6% CD45RA+ cells which are composed of slg+ B cells (approximately 0.4%), slg- CD4-CD8- cells (< 0.2%), and CD4+ CD8+ cells (< 0.2%). In some strains an additional CD45RA+ population, representing up to approximately 2% of all thymocytes, is present and has a CD4-CD8+ phenotype. It is this CD4-CD8+CD45RA+ subset which is responsible for the observed strain difference. In BALB/c mice, this additional population comprises approximately 90% of the CD45RA+ thymic cells. They are larger than the majority of thymocytes, with a size typical of mature, single positive cells (CD4+CD8- or CD4-CD8+). Further phenotyping for co-expression of other maturation markers showed them to be distinctive; they are CD3int-hi, i.e. as bright as other CD8 single positives, which are dimmer than CD4 single positives. In addition they are CD44hi, MEL-14dim and hi, Thy-1lo, HSAlo/-, and PNAlo, suggesting them to be amongst the most mature cells in the thymus. This was corroborated by their phenotypic similarity to CD45RA+ lymph node T cells. Furthermore, in BALB/c adult thymus sections, CD45RA+ cells are localized mainly in the medulla, consistent with a mature phenotype. Comparable with most mature thymocytes, cell cycle analysis revealed this subset to be composed of resting (G0/G1) cells. The CD4-CD8+CD45RA+ cells are amongst the most mature thymocytes and yet are indistinguishable from peripheral T cell counterparts; the possibilities that they are mature thymocytes due to exit the thymus, or that they may represent recirculating peripheral T cells, are discussed.