Using cDNA and genomic probes representing the alpha, beta, and gamma chain of the human T cell receptor genes, we have examined the structure and expression of these genes in 14 human leukemic T cell lines, representing different stages of thymic differentiation, and 15 functional human T cell clones. Rearrangement of the gamma and beta chain genes was found in all of the functional T cell clones and all but one (P30/OKUBO) thymic leukemia cell line; all of the lines that had rearrangement of the beta chain expressed beta mRNA. Expression of the alpha chain was found in all of the functional T cell clones examined, while rearrangement of the alpha chain gene, using currently available probes to the J region, could be shown in 10 of 13 functional clones. In contrast, expression of the alpha chain was found in 6 of 10 leukemic T cell lines, while rearrangement was found in six of these nine cell lines. Of the 14 leukemic cell lines studied for rearrangement of the alpha chain, rearrangement was found in six cases. The data obtained with the cell lines are consistent with an ordered rearrangement and expression of the gamma, beta, and alpha chains of the T cell antigen receptor (TcR) genes. The leukemic cell lines used in the present study have previously been characterized with regard to cell surface antigens and intracellular enzymes. Based on those results a scheme of thymic development was proposed. The developmental stages identified by those studies are not in complete agreement with stages of T cell development, as determined in the present study using molecular probes.