The growing safety problems about the use of bone morphogenetic protein 2 (BMP-2) is one of the recent issues that was improved by using low doses of BMP-2 with the support of other osteoinductive agents and/or using appropriate carriers. The aim of the present study is to investigate the effect of scaffold-based dual release system including melatonin (MEL) and BMP-2 loaded polylactic-co-glycolic acid (PLGA) microparticles on the osteogenic activity of pre-osteoblastic MC3T3-E1 cells. MEL and BMP-2 loaded microparticles were prepared by double emulsion solvent evaporation method in the average diameters of similar to 2 mu m and similar to 11 mu m, respectively and loaded into chitosan/hydroxyapatite (HAp) scaffolds. In vitro MC3T3-E1 culture studies were carried out comparatively with blank scaffolds, single (BMP-2 or MEL) releasing groups and dual (BMP-2 and MEL) releasing group. Microscopic observations and hematoxylin/eosin staining showed enhanced number of cells and dense ECM in dual release group. The expressions of differentiation markers, Runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) and also mineralization were higher in dual release group than that of the other groups. Our findings showed that BMP-2 at low doses (similar to 20 ng per scaffold) was sufficient in terms of osteogenic activity with controlled release systems where it was used in combination with MEL (similar to 10 mu g per scaffold).