Identification of the t(X;18)(p11.2;q11.2) that is associated with a high proportion of synovial sarcoma can be a useful diagnostic aid. The translocation results in fusion of the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene, two homologous genes within Xp11.2. Two-color interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction were assessed as approaches to identify the rearrangement in well characterized cases. The presence of the translocation, and the specific chromosome X gene disrupted, were inferred from the configuration of signals from chromosome-specific centromere probes, paints, and markers flanking each gene in preparations of interphase nuclei. Rearrangement was found in two cell lines and eight of nine tumor samples, including analysis of five touch imprints. This was consistent with cytogenetic data in four cases and reverse transcription polymerase chain reaction analysis using primers known to amplify both SYT-SSX1 and SYT-SSX2 transcripts. The transcripts were distinguished by restriction with LspI and SmaI. Contrary to previous suggestions, there was no obvious correlation between histological subtype and involvement of the SSX1 or SSX2 gene. These approaches could also be applied to the identification of tumor-free margins and metastatic disease.