Studies were conducted to determine the role that diffusion may play in the in vivo kinetics of the Escherichia coli periplasmic enzyme, alkaline phosphatase (AP, encoded by the genepho A). Passive diffusion of solutes, from solution into the periplasm, is thought to occur mainly through porins in the outer membrane. The outer membrane therefore serves as a diffusion barrier separating a population of periplasmic enzymes from bulk substrate. E. coli strains containing a plasmid with the pho A gene linked to the lac promoter were used in this study in order to vary the amount of enzyme per cell. Alkaline phosphatase assays were conducted with intact cells, and the substrate concentration at half-maximum velocity (normally the Km for the enzyme) was determined as a function of enzyme concentration per cell. The results showed that diffusion of substrate to the enzyme caused as much as a 1000-fold change in this parameter, compared to that of purified enzyme. This suggested that diffusion was the rate-limiting step of the enzymatic reaction in these cells. In agreement with this type of reaction, Eadie-Hofstee and Lineweaver-Burk plots were not linear. At their extremes, these plots represented two types of kinetics. At high substrate concentration, equilibrium of substrate between bulk solution and the periplasm was achieved, and the kinetic properties conformed to Michaelis-Menten. At low substrate concentrations, there were a large number of free (unbound) enzymes, and each substrate molecule that entered the periplasm, through the diffusion barrier, resulted in product formation. In the latter case, the periplasm behaved as a perfectly reactive vessel, and enzyme velocity increased in direct proportion to substrate concentration. This is a useful kinetic approach to estimate diffusion rates through the outer membrane. Diffusion rates can be valuable for investigating variation in expression and permeability of porins in the outer membrane. This study shows how, in vivo, the conditions required for Michaelis-Menten analysis of enzyme kinetics are not met and more complex behavior is observed.