We performed immunohistochemical analyses of 568 breast/cancer specimens using Ki-67, a monoclonal antibody specific for a nuclear antigen present in proliferating cells. The specimens were divided into three groups (I-III) according to the proportion of Ki-positive cells detected. These findings were compared with features of tumor extension as well as with certain prognostic variables. There was no detectable correlation between Ki-67 reactivity and either tumor size or node involvement. In contrast, a statistically significant correlation was found between Ki-67 reactivity and tumor grading, in that G-I tumors had small growth fractions, while a high proportion of G-III tumors exhibited strong (group III) Ki-67 positivity. When growth fractions were compared with biochemical receptor status, a significant difference was detected between tumors with negative and positive findings for receptors. The same co-variation was observed with respect to the overexpression of neu-protein P185, with most neu-positive carcinomas being strongly positive for Ki-67 (group III). In the relapse cases examined, there was a close correspondence between Ki-67 reactivity and the duration of the disease-free period. Long-term observation of patients with primary breast carcinoma revealed that, with regard to overall survival, the less reactive groups I and II differed significantly from group III. With respect to disease-free survival, no difference was detectable between Ki-groups II and III, but when these two groups together were compared with group I, a significant trend emerged. Similar results for both overall and disease-free survival were obtained for subgroups of pT2 and G-II carcinomas as well as for receptor expression.(ABSTRACT TRUNCATED AT 250 WORDS)