The recently discovered MLT/MALT1 gene is fused with the API2 gene in the t (11; 18)(q21; q21), which characterizes about one-third of MALT lymphomas. In order to screen for variant translocations and amplifications of MLT/MALT1, we have developed a novel, undirected two-color interphase fluorescence in situ hybridization (FISH) assay with two PAC clones flanking MLT/MALT1. This assay was applied to 108 marginal zone B-cell lymphomas (MZBCLs), including 72 extranodal MALT lymphomas, 17 nodal, and 19 splenic MZBCL. In 19 MALT lymphomas (26%), but in none of the nodal or splenic MZBCL, separated hybridization signals of the MLT/MALT1 flanking probes, were found. Further FISH analyses showed that 12 of these 19 cases displayed the classical t (11; 18) and the remaining seven cases revealed the novel t (14; 18)(q32; q21), involving the MLT/MALT1 and IGH genes. The frequency at which these translocations occurred varied significantly with the primary location of disease. The t (11; 18) was mainly detected in gastrointestinal MALT lymphomas, whereas the t (14; 18) occurred in MALT lymphomas of the parotid gland and the conjunctiva. Amplification of MLT/MALT1 was not observed in any of the lymphomas analyzed. We conclude that the translocations t (11; 18)(q21; q21) and t (14; 18)(q21; q32) represent the main structural aberrations involving MLT/MALT1 in MALT lymphomas, whereas true amplifications of MLT/MALT1 occur rarely in MZBCL.