We studied the effects of basal medium and human serum on the ex vivo expansion of CD56+/CD3natural killer cells (NK). We demonstrated that sorted NK cultured for 18 days with 10% human AB serum without accessory cells in a 2:1 DMEM/F12 basal medium expanded significantly greater (6.4 ± 0.9-fold, n = 14) than when cultured in standard RPMI 1640 basal medium (3.7 ± 0.67-fold, n = 16; p = 0.019). Supplementation of the DMEM/F12 mixture with 2-mercaptoethanol (2ME), ethanolamine, L-ascorbic acid, and sodium selenite significantly augmented NK proliferation (16.3 ± 2.5-fold, n = 11; p < 0.001) compared with DMEM/F12 without supplements. NK growth kinetics demonstrated that both the growth rate and the duration of exponential growth were increased by medium supplements. Addition of 2ME-containing supplements to cultures of NK with accessory cells augments NK proliferation, recruits NK progenitors, and has a serum-sparing effect. For the large-scale expansion of NK, we demonstrate a greater than 30-fold increase (n = 7) in the number of activated natural killer cells (ANK) derived from CD5/CD8-depleted PBMNC when cultured for 35 days in supplemented DMEM/F12 versus RPMI 1640 basal medium (197.6 ± 95.0-fold versus 6.3 ± 2.1-fold, respectively). Serum-free media (AIM-V and X-VIVO 10) were unable to support NK proliferation. These data suggest that utilizing 2:1 DMEM/F12 + 2ME-containing supplements instead of standard RPMI 1640 as a basal medium can increase the proliferation of cytotoxic NK while reducing the culture interval and the amount of serum needed. Replacing RPMI 1640 with supplemented DMEM/F12 may markedly increase the efficiency of large-scale ex vivo NK expansion necessary for clinical immunotherapy trials.