The inoculum age and density markedly influence the productivity and economics of bioprocesses. Some literature reports demonstrated the use of two-stage inocula for the production of surfactin. However, no systematic studies to elucidate the individual, cumulative and interactive effects of the important twin parameters, inoculum age and size on surfactin production, have been reported. Thus, a computer aided response surface modeling, optimization and analysis of the age and size of the two-stage inocula was carried out in batch reactor studies. The optimal values thus, obtained include primary inoculum age and size = 56 h and 5.5% (v/v), secondary inoculum age and size = 4.5 h and 9.5% (v/v), respectively. At the above optimal conditions, the model predicts a maximum relative surfactin concentration of 58-59 CMC-1, mainly due to a strong interaction between primary inoculum size and secondary inoculum age. The experimental verifications substantiated the model predictions by showing a maximum relative surfactin concentration of 58 CMC-1, which was found to be equivalent to about 1.3 g L-1 crude surfactin as estimated gravimetrically, thereby resulting in an improved production. © 2004 Elsevier B.V. All rights reserved.