ANKH (human homolog of progressive ankylosis) regulates inorganic pyrophosphate (PPi) transport. Dominant ANKH mutations were detected in at least five multiplex families with calcium pyrophosphate dihydrate crystal deposition disease (CPPPD). The objective of this study is to assess the functional consequences of one CPPDD-associated ANKH mutation (ΔE490) in chondrogenic ATDC5 cells. Stable ATDC5 transfectants bearing myc-tagged constructs of wild-type ANKH, mutant ANKH (ΔE490) and neo controls were generated. Upon ITS (insulin, transferrin and selenium) induction, expression of chondrocyte markers including alkaline phosphatase activity in the various transfectants was assessed. The ANKH ΔE490- transfectants had low alkaline phosphatase activities throughout ITS treatment due to lower TNAP protein expression and the presence of intracellular low-molecular-weight inhibitors. Our results suggest that the interplay of ANKH and TNAP activities is tightly regulated.