Two culture media were designed and optimized by statistical techniques for the growth of Escherichia coli K12 HB101 (pCG402) which expressed human tumour necrosis factor-beta (TNF-beta). Common compounds, such as MgSO4 and KH2PO4, were used to provide the bacteria with the necessary elements for biomass synthesis. For compounds not required for biomass synthesis, such as thiamine, or compounds used for both biomass synthesis and as an energy source, such as glucose, yield coefficients were used. In formulating the composition of the nutrients, carbon was chosen as the limiting substrate in both media. In the complex medium (CM), casein hydrolysate was selected to supply the two auxotrophic amino acids, proline and leucine. The optimal ratio of glucose to casein hydrolysate was determined to be 1:0.6 by using a centre composite design experiment. In the defined medium (DM), the concentrations of the three carbon sources, glucose, proline and leucine were based on their respective yield coefficients (Y-biomass/glucose : 0.5, Y-biomass/proline : 7, Y-biomass/leucine : 13.5). Shake flask experiments based on a fractional design were used to confirm that the two media were glucose limiting. Bacterial growth was improved in CM whereas DM gave higher TNF-beta expression. A 2-dm(3) fed-batch fermentation using CM was performed and a dry biomass concentration of 20 g dm(-3) was obtained with the expression of soluble TNF-beta being 20 mg g(-1) dry biomass. With this fed-batch system, a high biomass concentration and high expression of TNF-beta were achieved concomitantly.