PHOSPHO1, a new member of the haloacid dehalogenase superfamily, has recently been implicated in the mineralization process in both osteoblasts and chondrocytes. In this study we describe the identification of a novel, alternatively spliced PHOSPHO1 transcript (PHOSPHO1-3a). This transcript contains the three exons of the previously published variant, however exon 3 contains a retained, 127bp section of intron 2. This forms an in-frame start site, producing an open reading frame of 879bp and predicting a protein of 292 amino acids. The novel 40 amino acid N-terminal region of PHOSPHO1-3a contains a relatively strong secretory signal, however all three domains of the HAD superfamily are retained in exon 3. The expression of this splice variant was confirmed in both human and mouse osteoblast-like cells and also in the chondrogenic ATDC5 cell line. The data within this study indicate a possible function relating to chondrocyte differentiation/mineralization as with the previously published variant.