A panel of murine mAb against chicken TCR and associated molecules was used to study the ontogeny of T cells. The intrathymic maturation of the TCR-gamma delta, (TCR-1) and TCR-alpha beta (TCR-2) sublineages was the focus of these studies employing immunoperoxidase staining of tissue sections and immunofluorescence analysis of cell suspensions. The first CD3+ cells appeared in the thymus on embryonic day 9 (E9) when the CD3 Ag was restricted to the cytoplasm. In tissue sections, both TCR-1+ and TCR-2+ cells were observed on E12, whereas only the TCR-1 cells were identifiable by surface immunofluorescence. On the next day, when a discrete thymic medullary region was first recognizable, the TCR-1 cells were present in both cortex and medulla. Two days later (E15), TCR-1 cells were found in the spleen. Surface TCR-2+ cells did not appear until E14, began to migrate in to the medulla on E17, and appeared in the spleen on E19. The first TCR-1 cells thus move quickly through this maturational pathway, whereas TCR-2 cells undergo a prolonged developmental period in the cortex. While most TCR-1+ cells were CD4-CD8-, a minor subpopulation (5 to 15%) were CD4-CD8+, and less than 1% were CD4+CD8+. In contrast, immature TCR-2+ thymocytes in the cortex were predominantly CD4+CD8+, whereas cells expressing a higher density of the CD3/TCR-2 complex were either CD4+CD8- or CD4-CD8+ and were localized in the thymic medulla. In the medulla of the mature thymus, the TCR-1+ cells preferentially occupy the cortico-medullary junction and form small aggregates around vessels. TCR-2+ cells were less frequent in these areas of TCR-1 accumulation. The thymic ontogeny and, by implication, the selection of the receptor repertoire thus differs substantially for these two TCR isotypes.