Objectives To establish a simple, inexpensive and efficient technique for in vitro culture of monocyte-derived macrophages (MDM) from rhesus macaques of Chinese origin. Methods Peripheral blood of healthy rhesus macaques (Macaca mulatta) were obtained in heparinized vacutainer collection tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from blood by Ficoll gradient centrifugation. Serum was isolated from peripheral blood of the autologous animals. PBMCs were plated in 48-well-plate (3×10⁶ cells/well) or 96-well-plate (0.8×10⁶ cells/well) for 24 h. After removal of non-adherent cells from the culture, monocytes were cultured in RPMI 1640 supplemented with different proportions (2%, 4%, 8%, 10%) of autologous serum or fetal calf serum (FCS) for 7 days. To examine the biological function of MDM, lipopolysaccharide (LPS) was added to MDM culture to determine inflammatory cytokine production. Also, MDM cultures were tested for the susceptibility to simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) infections. Results The cell cultures with RPMI1640 containing 2% autologous serum yielded the best results with regard to macrophage morphology, the response to LPS stimulation and susceptibility to SIV or SHIV infection. The purity of adherent macrophages under condition of 2% autologous serum culture was higher than 96%. Conclusions RPMI 1640 with 2% autologous serum is suitable for culture in vitro of peripheral blood monocytes from rhesus macaques. This technique is simple, inexpensive, no need for growth factor and highly effective to obtain adherent and well differentiated macaque monocytes. Therefore, this method provides an important tool for culture of macaque AIDS viruses and for related immunological research.