This paper describes medium optimization for urease production by Aspergillus nigerPTCC5011 by one-factor-at-a-time and orthogonal array design methods. The one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. Among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. Subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. The effects of nutritional components for urease production by A. niger PTCC5011 in the first and second stages were in order of urea > NiSO(4) > sucrose > KH(2)PO(4) > K(2)HPO(4) > CaCl(2) > yeast extract > MgSO(4) and yeast extract > sucrose > K(2)HPO(4) > KH(2)PO(4) > urea > CaCl(2) > NiSO(4), respectively. The optimal concentrations of nutritional components for improved urease production were determined as 20 gl(-1) sucrose, 0.85 gl(-1) urea, 3.4 gl(-1) yeast extract, 0.03 gl(-1) NiSO(4)(.)6H(2)O, 0.5 gl(-1) MgSO(4)(.)7H(2)O, 0.04 gl(-1) CaG(2), 5.5 gl(-1) KH(2)PO(4) and 0.35 gl(-1) K(2)HPO(4). These results showed that urea, NiSO(4), yeast extract and sucrose had significant effect on urease production by A, niger PTCC5011. Tween 80 and MgSO(4) had negligible effect on urease production by this strain. The subsequent confirmation experiments determined the validity of the models. Maximum urease activity in optimized media by one-factor-at-a-time and orthogonal array methods were about 1.41 and 2.74 times greater than with the basal medium, respectively. (c) 2005 Elsevier Ltd. All rights reserved.