Pleomorphic adenoma (PA), a benign mixed salivary gland tumor, has been associated with abnormal karyotypes in up to 70% of cases, with nonrandom involvement of 8q12, the locus of the pleomorphic adenoma (PLAG1) gene. In this study, cytogenetics and fluorescence in situ hybridization (FISH) were used to investigate PLAG1 involvement in PA from seven patients. There were two males and five females ranging in age from 25 to 65 years. Samples of parotid gland tissue from the tumor sites, set up as solid tumor cultures, showed a normal karyotype in two cases [46,XY;46,XX] and cytogenetic abnormalities in five cases (71%). The abnormalities comprised one variant translocation [t(1;4;8)(p32;q35;q12)], two classic translocations [t(5;8)(p13;q12)], one novel deletion [del(12)(p11.2p12.1)], and a novel insertion [ins(9;8)(p22;q12q21.1)]. FISH was performed in all cases by using two probes from the RP11 library, flanking PLAG1; a sequence 1.48 megabases (Mb) upstream and another 2.27 Mb downstream, covering a total area of 3.8 Mb. The PLAG1 gene was intact and normally situated in four cases - the 46,XY, 46,XX, del(12p), and one t(5;8). PLAG1 was disrupted in three cases - one t(5;8), ins(9;8), and t(1;4;8). In addition, genomic instability was seen in two cases, one with PLAG1 amplification in the form of a homogeneously staining region, and the other in der(8) ring formation. The data provide further unique cases showing the complexity of PLAG1 gene rearrangements in PA.