BACKGROUND Immunocytochemical techniques are a potentially valuable diagnostic tool to support cytologic diagnosis in dogs. However, detailed studies of staining patterns and intensity in cytologic specimens of mesenchymal tumor types are lacking. OBJECTIVE The aim of this study was to evaluate commercially available antibodies against human proteins for use in the characterization of canine tumors of mesenchymal origin in cytologic samples. METHODS Immunocytochemical staining was performed on air-dried imprint specimens of biopsies obtained from 103 mesenchymal neoplasms and 14 metastatic lesions from 98 dogs. All specimens were stained with anti-cytokeratin AE1/AE3 and vimentin. Based on the histologic diagnosis, tumors of muscle, endothelial, histiocytic, and melanocytic origin also were stained with cell-specific antibodies. Staining intensity was subjectively graded and the percentage of positive tumor cells was estimated. RESULTS All mesenchymal tumors and metastases, with the exception of mesotheliomas, were vimentin-positive and cytokeratin-negative; mesotheliomas (n=6) were positive for both vimentin and cytokeratin. Tumors of muscle (n=5), endothelial (n=15), and histiocytic (n=18) origin stained moderately to strongly positive in a majority of tumor cells with desmin, von Willebrand factor, and lysozyme, respectively. Malignant melanomas (n=15) had variable staining and a variable percentage of positive cells with Melan-A and S100. CONCLUSIONS Our results indicate that immunocytochemical staining of canine cytologic specimens is a reliable and sensitive technique that may be of benefit for the differentiation of poorly differentiated mesenchymal tumors and metastases. Additional study is needed to assess the specificity of immunocytochemical stains in mesenchymal tumors.