Using a newly isolated cDNA clone encoding the TCR-delta gene and genomic probes, we have analyzed T cell receptor (TCR) delta gene rearrangement in 19 patients with T cell acute lymphoblastic leukemia (T-ALL) and 29 patients with B-precursor ALL. Five out of seven CD3- T-ALL and 4 of 12 CD3+ T-ALL showed bi-allelic rearrangements of the TCR-delta gene. In three CD3+ patients, a single allelic TCR-delta gene rearrangement was observed with rearrangement of the TCR-alpha gene on the other allele. In five CD3+ patients with bi-allelic rearrangements of the TCR-alpha gene, the TCR-delta gene locus was deleted. Transcription of the TCR-delta gene was also analyzed in six T-ALL. Five patients expressed TCR-delta transcripts. Only one T-ALL, presumably derived from the most immature T lineage cells, did not have TCR-delta transcripts, but expressed TCR-gamma and 1.0-kb truncated TCR-beta transcripts. In B-precursor ALL, 20 patients (69%) showed rearrangements of the TCR-delta gene. The frequency of TCR-delta gene rearrangement was higher than TCR-alpha (59%), gamma (52%), or beta (31%) genes. These findings suggest that TCR-alpha gene rearrangements may take place after rearrangements of the TCR-delta gene with concomitant deletion of rearranged TCR-delta genes in T cell differentiation. Among leukemic cells of B lineage, the TCR-delta gene is the earliest rearranging TCR gene, followed by TCR-gamma and beta gene rearrangements.