Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E. coli. Operational conditions for maximum production were determined with twelve parameters under consideration: the concentration of FeCl(3), induction at OD(578) (optical density measured at 578 nm), induction time and inoculum concentration. Initially, Plackett-Burman (PB) design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90x10(-3) U/ml by the best combinations of the physicochemical parameters at optimum levels of 0.12 mg/L FeCl(3), inoculum concentration of 2.10%, induction at OD(578) equal to 1.07, and with 6.05 h of induction.