The functional maturation process of medullary-type CD4^–CD8⁺ [CD8 single-positive (SP)] thymocytes remains largely uncharacterized. We describe a phenotypic analysis of CD8 SP medullary-type thymocytes and find a remarkable heterogeneity within this thymic cell population. While mature CD8⁺ T cells in the periphery are relatively homogenous (TCRαβ⁺CD3⁺Qa-2⁺ HSA^–3G11^–6C10^–CD69^–), CD8 SP medullary-type thymocytes contain discrete subpopulations that can be identified by differential expression of several cell-surface markers. We have identified at least six discrete subpopulations in the subset of TCRαβ⁺CD3⁺ CD8 SP cells in the thymus. According to the expressed phenotypes, a linear developmental pathway is predicted among these CD8 SP subpopulations as follows: 6C10⁺CD69⁺HSA^hi3G11⁺Qa-2^– → 6C10^–CD69⁺HSA^hi/int3G11⁺Qa-2^– → 6C10^–CD69^–HSA^int3G11⁺Qa-2^– → 6C10^–CD69^–HSA^lo3G11⁺Qa-2^– → 6C10^–CD69^–HSA^–/lo3G11^–Qa-2^– → 6C10^–CD69^–HSA^–/lo3G11^–Qa-2⁺. This study provides a framework for understanding CD8 SP T cell maturation in the thymic medulla.