AbstractMembranes of the autologous blood‐derived biomaterial platelet‐rich fibrin (PRF) were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, and subsequently incubated in calcium glycerophosphate (CaGP) solution to induce PRFs mineralization with calcium phosphate (CaP) to improve PRFs suitability as a material for bone replacement. Incorporated ALP retained its bioactivity and induced formation of CaP material within PRF membranes, as confirmed by SEM, EDS, FTIR, and von Kossa staining. The mass percentage attributable to CaP was quantified by lyophilization and measurement of the remaining mass fraction as well as by TGA. Cytocompatibility tests (LDH, MTT, and WST) with SAOS‐2 cells showed that mineralized PRF did not release substances detrimental to cell vitality. Live/dead staining and SEM showed that mineralized PRF was colonized by cells. The results show that hydrogel biomaterials such as PRF can be mineralized through functionalization with ALP. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 2012.