Elevated serum levels of the protein YKL-40 are associated with a poor prognosis in patients with solid and hematologic malignancies including breast cancer. The aim of this study was to develop a valid reproducible immunohistochemical method to visualize YKL-40 expression in normal breast tissue as well as in benign and malignant breast lesions. The presence of YKL-40 in breast tissue was verified by in situ hybridization and protein extraction procedures. An immunohistochemical method was developed and 4 different antibodies directed against YKL-40 were tested. Ten patients with normal breast tissue and benign breast lesions and 53 patients with localized breast carcinomas were analyzed immunohistochemically. The presence of YKL-40 in normal epithelial cells as well as in malignant tumor cells of the breast was established; however, a difference in staining intensity and staining pattern was observed. In normal breast tissue, a weak YKL-40 immunoreactivity was found in the cytoplasm of the epithelial cells with an additional strong dotlike staining between the nucleus and the gland lumen. In malignant lesions, 81% of the in situ carcinomas and 64% of the invasive carcinomas showed strong diffuse cytoplasmic YKL-40 immunoreactivity. No nuclear and membrane staining was found. A subpopulation of cells of macrophage morphology in normal breast tissue and in malignant lesions showed strong YKL-40 immunoreactivity.