Background/Aims: The possibility that substances penetrate gallstones and accumulate after stones have formed has not been examined. The specific aims of this study were to determine whether cholesterol gallstones are permeable and, if so, the effect of molecular weight on permeability. Methods: Cholesterol gallstones from patients with multiple stones were collected during surgery and incubated in fluorescein solution or in solutions of fluoresceinated albumin or immunoglobulin (lg) G. To determine egress from the stones, some stones were removed from the fluoresceinated solution after incubation and placed in bicarbonate buffer. The total area of the stone and the area of dye that had diffused into the calculi were calculated. To determine mass of penetrating IgG, stones were powdered after incubation, and IgG was measured by an enzyme-linked immunosorbent assay. Results: All substances penetrated stones. Although all compounds tested diffused back out of the stones when they were replaced in buffer, proteins did so more slowly than fluorescein. Conclusions: Substances of different molecular weights can diffuse into and out of cholesterol gallstones. These findings must be taken into account when considering the role of substances contained in stones on stone formation and growth.