The meta-cleavage pathway of Pseudomonas putida mt-2 was simulated using a biochemical systems simulation developed by Regan (1996). A non-competitive inhibition term for catechol-2,3-dioxygenase (C23O) by 2-OH-pent-2,4-dienaate (K-i = 150 mu M) was incorporated into the model. The simulation predicted steady state accumulation levels in the mu M range for metabolites pre-mete-cleavage, and in the mM range for metabolites post-meta-cleavage. The logarithmic gains L[V-i(-), X-j] and L[X-j(-), X-j] clearly indicated that the pathway was most sensitive to the concentration of the starting substrate, benzoate, and the first enzyme of the pathway, toluate-1,2-dioxygenase (TO). The simulation was validated experimentally; it was found that the amplification of TO increased the steady state flux from 0.024 to 0.091 (mmol/g cell dwt)/h. This resulted in an increased accumulation of a number of the pathway metabolites (intra- and extracellularly), especially cis-diol, 4-OH-2-oxovalerate, and 4-oxalocrotonate. Metabolic control analysis indicated that C23O was, in fact, the major controling enzymic step of the pathway with a scaled control coefficient of 0.83. The amplification of TO resulted in a shift of some of the control away From C23O, Catechol-[2,3-dioxygenase, however, remained as the major controling element of the pathway.