Purpose Chondroitin sulfate, which is less expensive and more inert than heparinoids, hyaluronan or pentosan polysulfate, has been introduced to restore the barrier function lost due to epithelial dysfunction in interstitial cystitis (IC). The binding of chondroitin sulfate to damaged bladder as a function of the range of pH seen in urine, its efficacy in restoring the bladder's permeability barrier, and the capacity of damaged bladder to bind chondroitin sulfate have not been determined previously. Methods Binding of chondroitin sulfate to bladder urothelium was investigated quantitatively using chondroitin sulfate highly labeled with Texas Red and quantitative fluorescence microscopy in a mouse model of acid damage of the urothelium. The efficacy of restoring the barrier function was determined using passage of intravesically instilled 86Rb, a potassium ion mimetic, through the urothelium into the bloodstream in a rat model of bladder damage. The binding capacity of acid-damaged bladder was determined by fluorometry. Results Chondroitin sulfate bound tightly and exclusively to the mouse bladder surface that had been damaged by acid but showed only minimal binding to undamaged bladder. There was no systematic variation with pH. The model showed some variability in the degree of damage induced. In rats, chondroitin sulfate instillation restored permeability to 86Rb to control levels. Binding was saturable at 0.67 ± 0.13 mg/cm2 of bladder surface. Conclusions Chondroitin sulfate binds preferentially to damaged urothelium and restores the impermeability barrier. This suggests that the GAG layer is a major contributor to the impermeability of bladder urothelium. As determined by the binding capacity, the dose applied to humans in Canada (400 mg per instillation) is sufficient to obtain maximum efficacy.