Objectives: To prove the biocompatibility of biomaterials applied in biomedical devices, in vitro testing is crucial to render a material fit for medical application. The material of choice for dental implants is commercially pure titanium (cp-Ti), while other materials such as zirconia and polyetheretherketone (PEEK) are considered highly promising due to their functional and esthetic properties. The aim of this study was to determine whether PEEK with defined mean surface roughness and composition could achieve results equal to titanium or zirconia. Materials and methods: Disks measuring 14 mm in diameter and 1 mm in thickness made from cp-Ti, yttria-stabilized zirconia (Y-TZP), and filled PEEK with a smooth surface finish were used for cell culture experiments. Human fetal osteoblasts (hFOB) were cultured in vitro on each material to observe changes after 1, 3, and 7 days regarding cell viability and lactate dehydrogenase (LDH) release. Additionally, mRNA expression of proliferative factors PCNA and Ki67 and cellular adhesion (vinculin mRNA expression and immunofluorescence staining) were analyzed after 3 days in the culture. Results: In hFOB cultures, adhesion and viability were decreased on PEEK platelets, while LDH release remained stable. No significant difference was observed in cp-Ti and Y-TZP when compared to the control. Conclusions: The performance of cp-Ti and Y-TZP was equal to the control in all tests. It seems that highly polished PEEK in this particular composition cannot be recommended for osseointegrated implant applications due to decreased osteoblast attachment. Further investigations are recommended, especially in surface structures optimized for osseointegration. Keywords: biocompatibility, osteoblasts, polyetheretherketone, PEEK, titanium, zirconia