Ideally, bone substitute materials would undergo cell-mediated degradation during the remodeling process of the host bone tissue while being replaced by newly formed bone. In an attempt to exploit the capacity of Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) to stimulate osteoclast-like cells formation, this study explored different loading methods for RANKL in injectable calcium phosphate cement (CPC) and the effect on release and biological activity. RANKL was loaded via the liquid phase of CPC by adsorption onto or incorporation into poly(lactic-co-glycolic acid) (PLGA) microspheres with two different morphologies (i.e., hollow and dense), which were subsequently embedded in CPC. As controls nonembedded PLGA-microspheres were used as well as plain CPC scaffolds with RANKL adsorbed onto the surface. RANKL release and activity were evaluated by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) and osteoclast-like cells formation in cell culture experiments. Results indicated that sustained release of active RANKL can be achieved upon RANKL adsorption to PLGA microspheres, whereas inactive RANKL was released from CPC-PLGA formulations with RANKL incorporated within the microspheres or within the liquid phase of the CPC. These results demonstrate that effective loading of RANKL in injectable CPC is only possible via adsorption to PLGA microspheres, which are subsequently embedded within the CPC-matrix. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.