Chromosomal aberrations with breakpoints in T-cell receptor (TCR) gene loci are recurrent in several T-cell malignancies. Although the importance of interphase cytogenetics has been extensively shown in B-cell lymphomas, hardly any molecular cytogenetic tools are available for recurrent changes in T-cell disorders. Thus, we have established fluorescence in situ hybridization (FISH)-based break-apart assays for the TCRA/D (14q11), TCRB (7q34) and TCRG (7p14) genes and the TCL cluster (14q32). The assays were validated in normal controls as well as in 43 T-cell malignancies with cytogenetically proven 14q11, 7q34-35 or 7p13-21 aberrations. Breakpoints in TCRA/D, TCRB and TCRG could be diagnosed by these assays in 32/33 T-cell neoplasms with chromosome 14q11, 3/6 with 7q34-35 and 1/7 with 7p13-21 alterations, respectively. Application of the new FISH assays to a series of 24 angioimmunoblastic and 12 cutaneous T-cell lymphomas confirmed the cytogenetic evidence of lack of breakpoints in the TCRA/D or TCRB locus. Simultaneous detection of TCRA/D or TCRB breaks was achieved in a multicolor approach, which was further combined with detection of the T-cell-specific CD3 antigen in a multicolor FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasm) assay. These new FISH and FICTION assays provide sensitive, rapid and accurate tools for the diagnosis and biological characterization of T-cell malignancies.