Studies were made on the kinetic properties of the active transport reaction of pro-line in cells of Escherichia coli. The kinetics of the entry of substrate gave a nonlinear, hyperbolic curve when the initial rates of uptake were plotted by the Line-weaver-Burk method. The two Km values thus obtained with cells grown in a synthetic medium were 0.44 and 40 μM, respectively. When the cells were grown in a semi-defined nutrient medium, the activity of proline uptake increased without alteration in its Km values. Formation of an intracellular pool of proline was determined as a function of the concentration of substrate. A 1000-fold intra- to extracellular concentration gradient was formed at substrate concentrations below 3 μM, followed by rapid overshooting of the proline pool. The proline uptake reaction was greatly affected by potassium and sodium ions and their effects in stimulating and inhibiting the uptake reaction were studied as function of their concentrations. The effects of various drugs inhibiting energy metabolism on the uptake reaction were analyzed. 2,4-Dinitrophenol and potassium cyanide mainly affected the uptake reaction, reducing the Vmax values, while iodoacetate and iodoacetamide affected it greatly, increasing the Km values. The effects of the drugs on the exchange reaction of proline transport were studied in detail. The exchange-entry reaction of substrate was facilitated by, and dependent on the concentration of substrate in the cells. The rate of exchange-entry was inhibited by 2,4-dinitrophenol and iodoacetate. A single, homogeneous transport carrier model with multi-binding sites for substrate was proposed to explain the active transport system of bacteria which gives curvilinear Lineweaver-Burk plots, and shows negative cooperativity.