Mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (sec) and dose-dependent decrease in intracellular pH (pH_in), indicating diffusion of fatty acids across the plasma membrane. pH_in returned to its initial value with a time course (min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with inhibitors phloretin or TriascinC had no effect on the rapid pH_in drop following addition of oleate but greatly suppressed pH_in recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or TriascinC, supporting the correlation between pH_in recovery and metabolism. We then used a dual fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH_in decreased rapidly and simultaneously, but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH_in drop or its slow recovery, but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.