To determine the utility of positive p53 protein immunostaining as an adjunct in the diagnosis of malignancy in pleural biopsy specimens, we reviewed 73 recently obtained pleural biopsy specimens that represented the typical range of diagnoses encountered in the evaluation of a proliferative pleural process. Immunohistochemistry was performed on paraffin sections of each biopsy specimen using a monoclonal antibody to the p53 suppressor gene product clone BP53-12 (BioGenex, San Ramon, CA) and a standard capillary gap (Microprobe, Fischer Scientific, Pittsburgh, PA) avidin-biotin complex technique with a citrate buffer antigen retrieval solution. Of the pleural biopsy specimens with unequivocal malignancy, 19 of 40 mesotheliomas and nine of 18 metastatic adenocarcinomas were immunopositive for p53 protein. All 13 of the biopsy specimens with reactive mesothelial hyperplasia or organizing pleuritis were negative. Two pleural biopsy specimens, which were interpreted as suspicious but inconclusive for malignancy, were positive for p53 protein and subsequent pathology specimens confirmed the presence of metastatic carcinoma in both of these biopsy specimens. Our findings suggest that p53 protein immunostaining is relatively sensitive and highly specific in differentiating reactive processes from primary or metastatic malignancies in histopathologically equivocal pleural biopsy specimens.