BACKGROUND: During aging or various diseases, pathologic mineralization may occur in joints or in the vascular wall. This is due to the deposition of phosphate (Pi)-containing crystals into the extracellular matrix of articular chondrocytes or vascular smooth muscle cells. The mineralization ability of chondrocytes and smooth muscle cells of other tissue has not been investigated. OBJECTIVE: In this context, our work seeks to study the response induced by Pi on cartilage and smooth muscle cells from tracheal origin. METHODS: We have established a dissection procedure to harvest and isolate chondrocyte and smooth muscle cells from adult mouse trachea. Both cell types were then exposed to different concentrations of Pi (1, 3 or 5 mM) up to 14 days. Mineralization was evaluated by alizarin red staining, which identifies calcium deposition. The expression of genes characterizing the phenotypic identity of the cells and involved in the mineralization process was assessed by RT-qPCR. RESULTS: Treatment of tracheal chondrocytes and smooth muscle cells with increasing concentrations of Pi (3 and 5 mM) induced mineralization as revealed by positive alizarin red staining as early as 7 days of culture. Moreover, gene expression analysis revealed profound phenotypic changes in both cell types and suggested they mineralize through TNAP-independent or - dependent mechanisms, respectively. CONCLUSIONS: Our data indicate that, comparably to articular chondrocytes or vascular smooth muscles, chondrocyte and smooth muscle cells from the trachea are prone to mineralize under high-phosphate conditions.