Reliable detection of clonal T cell receptor beta (TCRB) gene rearrangements is essential for clonality assessment of suspect T cell proliferations. Since no appropriate Southern blot probes were available, we developed a new set of optimized TCRB gene probes. The TCRBJ1 and TCRBJ2 probes are positioned just downstream of the Jbeta1 and Jbeta2 gene segments, respectively, and can be used for detection of both incomplete Dbeta-Jbeta and complete Vbeta-Jbeta rearrangements, whereas the TCRBD1U and TCRBD2U probes upstream of the Dbeta segments can be used to confirm incomplete Dbeta-Jbeta rearrangements. Less frequently occurring Vbeta-Dbeta rearrangements can easily be detected via the downstream TCRBD1 and TCRBD2 probes. Although both EcoRI and HindIII are appropriate restriction enzymes to be used in combination with all these probes, false-positivity due to partial digestion of the EcoRI site in the Jbeta2-Cbeta intron has to be excluded via the TCRBC probe. Application of the TCRB probes in a large series of nearly 200 T cell malignancies revealed clonal rearrangements in all immature and mature TCR alphabeta+ T cell malignancies, in the vast majority of TCR gammadelta+ T cell acute lymphoblastic leukemias (T-ALL) (approximately 95%) and even in most CD3- T-ALL (approximately 80%). TCRB gene rearrangement patterns differed between the various categories of T cell malignancies. An increased frequency of complete Vbeta-Jbeta1 rearrangements was observed in TCR alphabeta+ T-ALL as compared to CD3- and TCR gammadelta+ T-ALL (33% vs 16% and 11%, respectively), and also incompletely rearranged V-D, D-D, or D-J alleles in the beta2 region occurred more frequently in TCR alphabeta+ cases than in CD3- and TCR gammadelta+ T-ALL (27% vs 15% and 18%, respectively). Furthermore, in comparison to TCR alphabeta+ T-ALL, less Vbeta-Jbeta1 and more Vbeta-Jbeta2 rearrangements were detected in mature TCR alphabeta+ T cell malignancies. The occurrence of the different types of TCRB rearrangement patterns has implications for PCR-based clonality assessment and for PCR-based detection of minimal residual disease via TCRB gene analysis. For instance, focussing on the beta2 region of T-ALL will allow detection of rearrangements in 70%, 75%, and 90% of CD3-, TCR gammadelta+, and TCR alphabeta+ cases, respectively. Therefore the here-described results will facilitate the design of the most appropriate primer sets for PCR analysis of TCRB gene rearrangements at the DNA level.