Southern blot analysis of T-cell receptor (TCR)-delta gene rearrangements is useful for diagnostic studies on the clonality of lymphoproliferative diseases. We have developed 18 new TCR-delta gene probes by use of the polymerase chain reaction (PCR) techniques. Application of these probes for detailed analysis of the TCR-delta genes in normal control samples, 138 T-cell acute lymphoblastic leukemias (T-ALL), and 91 precursor B-ALL allowed us to determine the TCR-delta gene restriction map for five restriction enzymes, as well as the Southern blot restriction enzyme patterns of all theoretically possible TCR-delta gene rearrangements. Based on this information, it appeared that 97% of all 213 detected TCR-delta gene rearrangements in our series of ALL could be detected by use of the TCRDJ1 probe and that the majority (76%) of the 213 rearrangements could be identified precisely. In T-ALL, we found a strong preference for the complete rearrangements V delta 1-J delta 1 (33%), V delta 2-J delta 1 (10%), and V delta 3-J delta 1 (7%) and the incomplete rearrangement D delta 2- J delta 1 (11%). In precursor B-ALL, the majority of rearrangements consisted of V delta 2-D delta 3 (72%) and D delta 2-D delta 3 (10%). The junctional diversity of these 6 preferential TCR-delta rearrangements was analyzed and showed an extensive junctional insertion (approximately 30 nucleotides) for complete V delta-J delta rearrangements, whereas incomplete rearrangements had correspondingly smaller junctional regions. The detailed TCR-delta gene restriction map and probes presented here, in combination with the Southern blot patterns of TCR-delta gene rearrangements, are important for TCR-delta gene studies in ALL; all TCR-delta gene rearrangements can be detected and the majority can be identified precisely. Identification of rearrangements is a prerequisite for subsequent PCR analysis of TCR- delta gene junctional regions, eg, for detection of minimal residual disease during follow-up of ALL patients.