Amino acid utilization of mouse macrophage-like RAW264.7 cells was investigated. During the logarithmic growth stage, RAW264.7 cells grew very fast, with an approximate doubling time of 11 hours, in DMEM supplemented with 10% heat-inactivated fetal bovine serum. RAW264.7 cells consumed glutamine at the fastest rate, followed by serine, leucine, isoleucine, arginine, lysine, valine and other amino acids. When the cell density reached a critical threshold level, cells began to suffer non-apoptotic cell death characterized by mitochondrial damage (revealed by transmission electron microscopy) and a smear pattern of DNA fragmentation (revealed by agarose gel electrophoresis). At this point, glutamine, serine and glucose in the medium were almost completely exhausted, whereas other amino acids remained at more than 40% of their initial concentrations. Based on these data, it is recommended that glutamine, serine and glucose should be supplemented for the long culture of RAW264.7 cells.