A chemically defined media was developed for growing Agrobacterium tumefaciens at large scale for commercial production of recombinant proteins by transient expression in plants. Design of experiments was used to identify major and secondary effects of ten media components: sucrose, ammonium sulfate ((NH4)(2)SO4), magnesium sulfate heptahydrate (MgSO4*7H(2)O), calcium chloride dihydrate (CaCl2*2H(2)O), iron (II) sulfate heptahydrate (FeSO4*7H(2)O), manganese (II) sulfate monohydrate (MnSO4*H2O), zinc sulfate heptahydrate (ZnSO4*7H(2)O), sodium chloride (NaCl), potassium chloride (KCl) and a sodium/potassium phosphate buffer (Na2HPO4/KH2PO4). Calcium and zinc were found to have no detectable impact on biomass concentration or transient expression level, and concentrations of the other components that maximized final biomass concentration were determined. The maximum specific growth rate of Agrobacterium strain C58C1 pTFS40 in this media was 0.33 +/- 0.01 h(-1) and the final biomass concentration after 26 h of batch growth in shake flasks was 2.6 g dry cell weight/L. Transient expression levels of the reporter protein GUS following infiltration of a recombinant Agrobacterium strain C58C1 into N. benthamiana were comparable when the strain was grown in the defined media, Lysogeny Broth (LB) media, or yeast extract-peptone (YEP) media. In LB and YEP media, free amino acid concentration was measured at three points over the course of batch growth of Agrobacterium strain C58C1 pTFS40; results indicated that L-serine and L-asparagine were depleted from the media first, followed by L-alanine and L-glutamic acid. (C) 2017 American Institute of Chemical Engineers