Human nerve growth factor (hNGF) and brain-derived neurotrophic factor (BDNF) were synthesized in a cell-free protein synthesis system using the S30 extract from Escherichia coli. Although 30 mu g of hNGF or 20 mu g of BDNF was synthesized from 1 ml of the reaction mixture in 2 h at 37 degrees C, the biological activities of these factors were very low because three disulfide linkages were not formed. However, when dialysis or salting-out treatment was carried out after protein synthesis, the biological activity of hNGF increased from 15% to 55% and that of BDNF from 40% to 60%. Cell-free protein synthesis followed by dialysis or salting-out should prove very useful in obtaining biologically active recombinant proteins.