The co-precipitation method followed by ultrasound and heat treatment is a common way to prepare below 100 nm sized hydroxyapatite nanoparticles for biomedical studies and applications. The size and pH value of the obtained calcium phosphate nanoparticles in aqueous sol have a strong impact on the interactions with cells and tissue. The physical and chemical properties of material samples for in vitro and in vivo studies are often assumed to remain constant from the time after fabrication to the actual use. Only little attention is paid to eventual changes of the material over time or due to the different in vitro conditions. In this study, the physical and chemical transformation of calcium phosphate nanoparticles after preparation and in vitro was investigated. As the result showed, dispersed nano sized amorphous calcium phosphate precipitation as well as crystallized hydroxyapatite nanoparticles continue to crystallize even when kept at 4 ℃ leading to declining pH values and particle sizes. Due to the pH buffer in the medium the pH value of the cell culture remained stable after adding 20% nanoparticle sol in vitro. However, hydroxyapatite nanoparticles immediately became unstable in the presents of cell culture medium. The resulting loose agglomerations showed a size of above 500 nm. The co-precipitation method followed by ultrasound and heat treatment is a common way to prepare below 100 nm sized hydroxyapatite nanoparticles for biomedical studies and applications. The size and pH value of the obtained calcium phosphate nanoparticles in aqueous sol have a strong impact on the interactions with physical and chemical properties of material samples for in vitro and in vivo studies are often assumed to remain constant from the time after fabrication to the actual use. Only little attention is paid to eventual changes of the material over time or due to the different in vitro conditions. In this study, the physical and chemical transformation of calcium phosphate nanoparticles after preparation and in vitro was investigated. As the result showed, dispersed nano sized amorphous calcium phosphate precipitation as well as crystallized hydroxyapatite nanoparticles continue to crystallize even when kept at 4 ℃ leading to declining pH values ​​and parti Due to the pH buffer in the medium pH value of the cell culture remained stable after adding 20% ​​nanoparticle sol in vitro. However, hydroxyapatite nanoparticles immediately became unstable in the presents of cell culture medium. The resulting loose agglomerations showed a size of above 500 nm.