Thymocyte heterogeneity has been studied by the use of two cell surface markers--the Ly-6.2 alloantigen and peanut agglutinin (PNA) receptor. The previously described distribution of Ly-6 on corticoresistant and PNA binding to corticosensitive thymocytes has been confirmed. Double immunofluorescence labeling experiments in combination with techniques of physical enrichment for "mature" thymocytes have resulted in the identification of a novel subset of T cells carrying both the Ly-6 antigen and PNA receptors. Purification by fluorescence cell sorting of corticosteroid-treated thymocytes, labeled with monoclonal anti-Ly-2.2 antiserum, for phenotypically mature Ly-1+23-lymphocytes causes a concomitant enrichment in the proportion of Ly-6-positive cells, which lack the PNA receptor. The implications of these findings for the maturational pathways within the thymus are discussed.