Multilayered coatings composed of mixtures of HA and P2O5-based bioactive glasses are of potential clinical benefit in orthopaedic and dental surgery. Pre-immersion of these materials has been reported to further enhance their efficacy in vivo, although the precise biological effects of this treatment are not yet known. In this study we have therefore prepared double-layer plasma-sprayed coatings and evaluated the effects of pre-immersion on the growth and function of human osteosarcoma cells in vitro, using the MTT assay and flow cytometry analysis, respectively. The results showed that the increase in numbers of viable cells was the same or elevated following incubation on the pre-immersed HA and glass-reinforced HA coatings compared with the non-immersed materials. In addition, the expression of bone sialoprotein and fibronectin, two key connective tissue antigens, was up-regulated in cultures grown on the pre-immersed surfaces compared with the non-treated materials. Moreover, cell numbers and antigen expression both improved as the proportion of glass increased, particularly in the pre-immersed samples. Our findings thus suggest that the immersion treatment of these materials appeared to improve the response of these bone-like cells.