Isolated rat enterocytes, both normal and those de-energized with rotenone, were used to study the energy dependence of membrane and intracellular intestinal riboflavin transport in vitro. Membrane and intracellular transport were investigated by using short (3 min) and long (20 min) incubation times, respectively. For both types of cells and incubation times, [3H]-riboflavin uptake presented a saturable component prevailing at physiologic intraluminal concentrations. At 3 min incubation, saturable [3H]-riboflavin transport was apparently an energy-independent process with high affinity and low capacity. Values of the saturable component and its apparent constants, Km and Jmax, did not differ in normal and de-energized enterocytes. At 20 min incubation, saturable [3H]-riboflavin transport was a strictly energy-dependent process in which values of the saturable component were significantly greater in normal than in de-energized enterocytes. Km values did not differ in the two types of cells and were unmodified over 3 min, whereas in normal enterocytes, Jmax at 20 min [6.25 +/- 0.2 pmol/(mg protein. 20 min)] was significantly greater than at 3 min [2.67 +/- 0.33 pmol/(mg protein. 3 min)] and compared with de-energized enterocytes at 20 min [2.54 +/- 0.16 pmol/(mg protein. 20 min)]. Both membrane and intracellular events were inhibited by unlabeled riboflavin and analogs, which are good substrates for flavokinase, thus demonstrating the paramount role of this enzyme in riboflavin intestinal transport.