Sixty-nine cases of clinical Stage I non-seminomatous germ cell tumors (NSGCT) of the testis were immunostained for the protein product of the p53 tumor suppressor gene using a microwave-based antigen retrieval method. It was assumed that the immunohistochemical detection of the p53 protein corresponded to a point mutation in the p53 gene, the wild-type p53 protein turning over too rapidly to be detected by routine immunohistochemical techniques. The results of p53 staining were then compared with the results, on the same paraffin tissue blocks, of S-phase analysis, as determined by flow cytometry, and the percentage of neoplastic cells exhibiting immunohistochemical positivity for proliferating cell nuclear antigen (PCNA). Thirty-four of 69 (49%) of the clinical Stage I NSGCT exhibited p53-positivity as strong, but focal, intranuclear positivity. Both the mean total S-phase and the mean percentage of PCNA-positive neoplastic cells were significantly higher in the p53-positive cases (27.8% and 89.6%, respectively) compared with the p53-negative cases (17.6% and 66.1%, respectively). Stratification of cases into high (> or = 76%) and low categories for PCNA values correlated significantly (P < 0.0005) with p53-positivity and negativity, respectively, by chi 2 analysis. The positive association of p53 protein expression with higher proliferative indices in NSGCT of the testis is consistent with the observation of p53 mutations correlating with markers of increased tumor aggressiveness in other types of neoplasia.(ABSTRACT TRUNCATED AT 250 WORDS)