Abstract Thymic involution is likely to be a significant factor in the alteration of peripheral T lymphocyte function with age. The process of thymic involution involves the progressive loss of normal organ architecture and cellular composition, and a significant reduction in the output of mature T lymphocytes. The present study assesses the impact of thymic involution on the T cell differentiation process by quantitating the number and percent representation of various phenotypically distinguishable T cell developmental intermediates in C57BL/6 mice of various ages. The results suggest that several distinct sites in the developmental sequence are impacted by aging. By middle-age (14–17 months), significant perturbations in the frequencies of several CD4 −CD8 − (DN) subpopulations have occurred. These include a shift towards an increased percentage of Pgp-1 + IL-2R − DN cells, the earliest thymic progenitors, and a decreased percentage and total number of Pgp-1 − IL-2R + DN cells. Furthermore there is a threefold increase in the percentage of DN cells which express CD3 (from 16.6% to 45.5%) which occurs between 4 and 14 months of age. By 24–27 months of age, the percentage of the total DN population increases two- to threefold over that of young (2–3 months) animals, while the fraction of CD4 +CD8 + (DP) is significantly reduced. These alterations are consistent with the possibility that thymic involution results in one or more ‘developmental’ blocks which limit key differentiative transitions within the DN population, and furthermore, the marked increase in the frequency of DN cells displaying CD3 argues that an alternative T cell differentiation pathway plays an increasingly significant role with advancing age.