Kinetic, metabolic, and physical parameters of long and medium chain fatty acid transport by Escherichia coli K12 were determined. Uptake of long chain fatty acids (C11-C18:1) mediated by the fadL gene involves concentrative transport. Evidence for this is as follows: (i) characteristic Ki and Vmax values were obtained for long chain fatty acids, (ii) long chain fatty acid transport was inhibited by energy inhibitors, (iii) long chain fatty acids were concentrated 10-fold inside the cell against a concentration gradient, (iv) efflux of transported long chain fatty acids did not occur, and (v) an energy of activation of 11.72 kcal mol-1 and Q10 of 2.3 were obtained for long chain fatty acid transport. The fadL gene product shows some activity with medium chain fatty acids (C7-C10) as well. Medium chain fatty acids also appear to enter the cell by simple diffusion since: (i) medium chain fatty acid transport by fadL strains is not saturable under our assay conditions, (ii) fadL strains do not concentrate medium chain fatty acids against a concentration gradient, and (iii) medium chain fatty acids are available for efflux in fadL strains. Physical parameters of long and medium chain fatty acid transport are also reported. These results present evidence for separate mechanisms of long and medium chain fatty acid transport in E. coli.