The full length cDNA of rat alkaline phosphatase (AP) was placed under the control of the SV40 early promoter. This plasmid was transfected by the calcium phosphate method into AP negative ROS 25/1 cells. Ten clones with AP specific activities ranging between 0.1–2 μmole/min/mg were isolated by cotransfection with the plasmid pSV2Neo, which renders the cells resistant to the antibiotic G418. Two clones with different AP specific activities: C (0.01 μmole/min/mg) and S (2.0 μmole/min/mg) and the osteoblastic ROS 17/2.8 cells (2.0 μmole/min/mg), were examined for their ability to mineralize. In vitro mineralization was tested by culturing cells in α-MEM containing 10% fetal bovine serum and 50 μg/ml ascorbate in the presence or absence of 10 mM β-glycerophosphate. Mineralized deposits were observed in all cultures of the S clone and ROS 17/2.8 cells in the presence of β-glycerophosphate, but not in C clones. Measurement of calcium and phosphorus levels in cells correlated with AP levels of transfected cells. However, extent of mineral accumulation in the transfected ROS 25/1 cells differ from the osteogenic ROS 17/2.8 cells. This finding indicates that high levels of AP may be a necessary constituent for the mineralization process together with other factors yet to be identified.