Rabbit spleen cells cultured in medium 199 with 20% fetal bovine serum or 20% autologous serum showed no measurable supernatant factor VIII (AHF) activity. In acellular control tissue culture vials containing medium 199, 20% horse serum, and 20% autologous plasma, activity averaged 29% at the beginning of incubation and 10% after 24 hours. In all instances, activity decreased to <1% by the end of the second day. In comparison, when initially heterogeneous rabbit spleen cell populations were cultured in medium 199, 20% horse serum, and 20% autologous plasma, a ralatively homogeneous macrophage culture was achieved by the third day, and measurable AHF levels were maintained for as long as nine days. Persistent AHF levels found in cultures but not in controls are interpreted as due to de novo synthesis of AHF by macrophages.