Definitive diagnosis of malignant mesothelioma in small specimens can be extremely difficult based on morphology alone. Homozygous deletion of 9p21, the locus harboring the p16 gene, has been reported as the most common genetic alteration in malignant mesotheliomas. Recent studies demonstrated that this alteration may be useful for differentiating benign from malignant mesothelial proliferations in cytology specimens. The aim of this study was to evaluate the diagnostic utility of homozygous deletion of 9p21 assessed by fluorescence in situ hybridization (FISH) in mesothelial proliferations involving serosal surfaces in paraffin-embedded tissue. p16 protein immunoexpression was also explored as a potential diagnostic aid. FISH analysis demonstrated homozygous deletion of the 9p21 locus in 35 of 52 cases (67%) of pleural mesothelioma and in 5 of 20 cases of peritoneal mesothelioma (25%) (P<0.005). None of 40 cases of reactive pleural mesothelial proliferations showed p16 deletion (P<0.005). Loss of immunoexpression of p16 was observed in 71% of the peritoneal mesotheliomas, 40% of the pleural malignant mesotheliomas and 15% of the reactive mesothelial cells. Homozygous deletion did not correlate with p16 protein expression in any of the studied groups. Our study suggests that 9p21 homozygous deletion assessed by FISH on paraffin-embedded tissue may be helpful for differentiating between malignant mesotheliomas and reactive mesothelial proliferations. A discrepancy between p16 protein expression and homozygous deletion suggests that other molecular mechanisms may play a role in p16 protein expression in mesothelial proliferations.