Two distinct antibodies raised against quail thymocytes were used in an indirect immunofluorescence assay to analyze the expression of differentiation antigens on T lineage cells in embryonic and newly hatched quail, (a) A rabbit serum, anti-AT-65, that has been previously shown to immunoprecipitate a protein of apparently 65-70 kDa from quail and chick thymocytes, reacts with both thymic and peripheral T cells. The polyspecificity of this pan-avian T cell reagent can be dissected by absorption and includes reactivities against two distinct antigenic entities. One of these is restricted to thymocytes whereas the other is also expressed on peripheral T cells, (b) A monoclonal antibody, a-TD1, defines the population of cortical thymocytes and does not react with peripheral T cells. Binding assays during the embryonic development of the T lineage reveal that specific surface markers are expressed 36 h following the seeding of lymphoid precursor cells to the thymic rudiment, suggesting their early commitment in this lymphoid microenvironment. T cells are also shown to be exported precociously to the embryonic splenic compartment.