S100 protein A8 and A9 naturally form a stable heterocomplex. Recently, we have proved that S100A9 overexpression in various adenocarcinomas is associated with poor tumor differentiation. In this study, we examined the relationship between the expression of each protein and the pathological parameters that reflect the aggressiveness of carcinoma, in invasive ductal carcinoma (IDC) of the breast. Serial paraffin-embedded tissue sections from 101 IDC cases were immunostained with respective monoclonal antibodies, and the results were as follows: 1) A positive correlation of immunoreactivity between S100A8 and S100A9 was noticed (r=0.873 and P < 0.0001); 2) The percentage of S100A9- positive tumor cells was higher than that of S100A8-positive tumor cells (P < 0.001), and S100A8 alone was not detected in any case; 3) Overlap between S100A8 and S100A9 staining patterns was found in the corresponding tissue areas, but S100A9 positivity was also observed in S100A8-negative tumor cells; 4) The immunopositivity for each protein also correlated with the mitotic activity, MIB-1 index, HER2 overexpression, node metastasis, and poor pT categories and pStage (P < 0.05); 5) Co-expression of both proteins was associated with poor tumor differentiation, vessel invasion, node metastasis, and poor pStage (P < 0.05). Furthermore, co-expression of the proteins was also observed in MCF-7 cells, and it was suggested that the immunolocalization is related with cell cycle. Our conclusions are as follows: 1) It is suggested that S100A8 is S100A9-dependently expressed and acquires the protein stability by S100A8/A9 heterocomplex formation; 2) S100A8 and S100A9 overexpression should be considered marker of poor prognosis in IDC.