When adenine (Ade) was added to basal culture medium at 12 It after inoculation, antivirus activity and specific activity of interferon-alpha2a (IFN-alpha2a) were increased. Expression of IFN-alpha2a was realized when a low residual glucose concentration was maintained during the mid-and late-phase of cultivation. In addition, biological activity and specific activity of IFN-a2a were increased by more than 100% if the ratio of glucose and sucrose in the basal medium was 1:0.1. The addition of glutamate also led to the intensive enhancement of the expression level of IFN-alpha2a. The initial pH of the basal medium proved to be crucial to the expression level. When the above optimal cultivation condition obtained in the shake flask was applied to the fed-batch culture using a 2.61 jar fermenter, human IFN-alpha2a biological activity reached 1.3 x 10(7) IU/ml, which was four times that of the control. (C) 2003 Elsevier Ltd. All rights reserved.