To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.