Protein kinase C (PKC) eta is a PKC isoform whose upregulation is associated with differentiation in many epithelial tissues, including the rat mammary gland. The purpose of this study was to examine whether PKC eta is altered, in expression or localization, in human breast cancer. Paraffin sections of 49 in situ breast lesions, 29 invasive breast tumors, and nine normal breast biopsies were examined for PKC eta expression by immunohistochemistry. Adjacent regions of normal epithelium, and in situ lesions that were present adjacent to invasive lesions were also analyzed. In normal epithelium, regardless of the presence of adjacent in situ or invasive lesions, PKC eta was present in the cytoplasm of the luminal epithelium, and increased in areas of normal lobular development, similar to normal rat mammary gland. PKC eta staining intensity was homogeneous in normal lobules, but heterogeneous in in situ and invasive lesions, being focally increased in cells with aberrant nuclear morphology. In situ lesions were similar to adjacent normal epithelium in average staining intensity, regardless of whether invasion was also present. However, the invasive lesions themselves were significantly decreased in staining intensity compared to adjacent in situ lesions. In addition, 75% of invasive breast cancer lesions showed decreased staining relative to adjacent normal epithelium, compared to 37% of in situ lesions. The invasive tumors which possessed high PKC eta staining were associated with positive lymph node status. These results demonstrate that quantitative and qualitative alterations in PKC eta occur in human breast cancers.