Nucleolar antigen PI 20 is detected in rapidly proliferating cells but not in normal resting cells or in many benign and slowly growing malignant tumors. The objective of the study was to determine whether the expression of PI 20 in breast cancer correlated with histopathological or biological properties associated with prognosis. In this retrospective study, 120 primary breast tumors were analyzed for PI 20; 114 of these tumors were also stained for the erbB-2 protein. Immunopositive staining was correlated with patient survival, nodal status, estrogen receptor levels, and number of mitoses. Sixty-nine % (83 of 120) of the tumors were positive for PI 20; 25% (28 of 114) stained positively for erbB-2. Of the 28 erbB-2 positive tumors 26 were also positive for the PI 20 protein. Forty-six % (55 of 120) of the specimens were from patients who later died from recurrent breast cancer; PI 20 was detected in 89% (49 of 55) of these specimens. In 52% of the survivors the PI 20 protein was also expressed. PI 20 negative tumors were highly correlative with survival (P = 0.0001); 84% (32 of 37) of patients with P120 negative tumors survived more than 7 years without evidence of recurrent disease. Multivariate analysis showed that the worst prognosis was for patients who had tumor positive nodes and expressed PI 20 (P = 0.0001); death occurred in 73% (30 of 41) of these patients. For the node negative patients who did not express PI 20, 5-year survival was 90% (19 of 21 patients); 5-year survival for the node negative patients who expressed P120 was significantly less (67%; 28 of 42 patients). Patients with P120 negative tumors had a good prognosis, irrespective of their nodal status. In this group, survival of node negative patients was 86% (18 of 21) and for those with positive nodes survival was 82% (13 of 16). A poor prognosis was found for patients with intense erbB-2 stained tumors (5 of 7 patients died). Weak staining of erbB-2 tumors (21 specimens) was not correlated with patient survival. Compared to PI 20 negative tumors, PI 20 positive tumors had greater numbers of mitoses (9.06 versus 6.65) and an almost 2-fold increase in the occurrence of positive nodes (one of every 4.67 versus one of every 8.81). The number of PI 20 positive tumors was greater in estrogen receptor positive tumors (75%) than in estrogen negative tumors (54%). These studies suggest that antigen PI 20 may be a prognostic marker in breast cancer and could be used along with other parameters such as nodal status to assess the prognostic outcome of breast cancer patients.