Activated keratinocytes play a key role in the cutaneous immune system by their interactions with other cell types through the production of cytokines with both autocrine and paracrine activity. But there is little knowledge about epidermal cytokines in the dog. In this study, cultured canine keratinocytes were stimulated by human recombinant interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) and cell supernatants were tested for tumour necrosis factor alpha (TNF-alpha) concentration using a cell viability assay on a murine cell line. We show that IFN-gamma in combination with LPS significantly increases TNF-alpha secretion by canine keratinocytes. The best stimulations were obtained using confluent cultures and the association of IFN-gamma (400 ng/ml) and LPS (40 microg/ml). The experimental protocol we describe represents a new method for studying keratinocyte activation and its modulation in the dog. We provide an example of application of our method: the study of the effects of different monosaccharides on canine keratinocyte activation.