While all thymocytes express CD45, only a small fraction (less than 3% in mice) bear the high molecular weight isoform, CD45RA. It has been suggested that these cells alone constitute the generative thymocyte lineage and should, therefore, occur within every ontogenic subset. To test this, we determined CD45RA expression among thymocyte subsets defined by CD4 and CD8. In some strains, exemplified by C57BL/Icrf, very few (less than 0.2%) T thymocytes expressed CD45RA and were mostly CD4-CD8- or CD4+CD8+, inconsistent with them constituting the generative lineage. In others, exemplified by BALB/c, CD45RA was expressed on up to 3% of T thymocytes, which were mostly CD4-CD8+. The limited occurrence of CD4-CD8+CD45RA+ thymocytes suggests that they are a nonconstitutive subset playing a role in the thymus of only some strains. Their occurrence correlates with that of Mtv proviruses within the mouse genome; however, their T cell receptor V beta repertoire is diverse, suggesting they are not uniquely selected by Mtv superantigens. We propose that they may be mature CD8 T cells, possibly responsible for introducing viral superantigens into the thymus.