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Casein phosphopeptides (CPPs) have been shown to enhance calcium solubility and to increase the calcification by in vitro analyses. The aim of our study was to investigate the effects of four selected casein peptides, which differ in the number of phosphorylated serines, on osteoblast mineralization in vitro. The chosen peptides, related to different casein genetic variants, were obtained by chemical synthesis and tested on murine osteoblast cell line (MC3T3-E1). Our results suggest that the distinct peptides in protein hydrolysates may differentially affect calcium deposition in the extracellular matrix and that the genetic variation within the considered peptides is involved in their differential effect.