Murine bone marrow derived macrophages (BMM) are valuable tools to investigate macrophage functions such as cytokine production and bactericidal activities from different strains of mice. In most studies BMM are generated and characterised using cell culture systems with fetal calf serum (FCS) as an essential supplement. Since serum contains varying amounts of undefined components influencing the maturation and polarisation process of BMM there is a need for a more standardised methodology. The aim of the present study was to establish a cell culture system for the generation of murine BMM under standardised serum free conditions. The use of a newly developed compositionally defined serum supplement enabled us to gain mature BMM from BALB/c and C57BL/6 mice expressing the myeloid marker F4/80, CD11b and MOMA-2. Under these serum-free conditions LPS and IFN-gamma stimulated C57BL/6 BMM released more IL-12 and nitric oxide (NO) compared to BALB/c BMM whereas the latter cells produced higher levels of IL-10 and MCP-1 after LPS stimulation. Serum-free generated C57BL/6 BMM showed enhanced bactericidal activity against the Gram-negative rod Burkholderia pseudomallei compared to BALB/c BMM. In conclusion the serum-free generation of BMM described in this study will assure more standardised and reproducible conditions for the future characterisation of murine BMM.