The walls of isolated upper Malpighian tubules of the insect, Rhodnius prolixus, are much more permeable to small relatively unchanged solutes (ethanol, xylose, and mannitol) than to larger or more charged solutes (acetate, glycine, tyrosine, and inulin). The more permeable solutes rapidly reach concentrations in the tubule cells equivalent to their concentrations in the bathing medium; the less permeable solutes do not penetrate into the cells. The time course of accumulation of permeable solute in the cells matches the time course of the appearance of solute in the lumen. Substances injected into the hemolymph of fed R. prolixus appear in the urine at concentrations predictable from the permeability of in vitro tubules, supporting the idea that the in vitro permeability of the tubules is representative of their properties in the intact insect. It is suggested that the rapid transcellular penetration of small solutes through the Malpighian tubules reflects the large areas of cell membrane. The area of cell membrane exceeds that of the paracellular clefts by a factor of 10(5).