Medium composition for transglutaminase (MTGase) production by Bacillus circulans BL32, a recently isolated strain from the Amazon basin, was optimized using a stepwise strategy. Effects of carbon, nitrogen and mineral sources were investigated by screening experiments. Plackett-Burman (P-B) statistical design was applied to find the key ingredients for the best medium composition among glycerol, sucrose, peptone, tryptone, Na2HPO4, MgSO(4)(.)7H(2)O and FeSO(4)(.)7H(2)O. Results indicated that sucrose, negatively, and peptone, positively, have significant effects on MTGase production, with confidence levels above 90%. Central composite design (CCD) was used to determine the optimal concentrations of these two components and the experimental results were fitted with a second-order polynomial model at 90% level (P < 0.10). Under the proposed optimized conditions, the model predicted the MTGase activity of 0.280 U/mL, very closely matching experimental activities of 0.306 U/mL. (c) 2005 Elsevier Ltd. All rights reserved.