The purpose of this study was to investigate the effect of the total degree of substitution (TDS) on the colligative properties of substituted cyclodextrins (CDs; i.e., b-and g-cyclodextrins substituted with sulfobutylether or hydroxypropyl groups) in solution. The osmotic pressure (OP) of aqueous CD solutions was determined over the concentration range 100ยฑ350 mmol/kg using vapor pressure osmometry. A chloride ion-selective electrode was used to help determine the possible mechanism for any CDยฑwater interactions that might occur by measuring the chloride-ion activity of a sodium chloride solution containing various CDs. Titration microcalorimetry was used to verify that sodium chloride did not interact with the CDs, thereby permitting the chloride ion activity to be utilized as an indicator of any possible CDยฑwater interaction. The results indicate that CDs with different total degrees of substitution (TDS) exhibit different colligative properties in solution. It was observed that increases in the degree of substitution of the CD produced an increased OP. All substituted CDs exhibited an OP above their theoretical values, whereas the OP of the unsubstituted g-CD was below its expected value, likely due to selfassociation. The results obtained with the chloride-ion selective electrode did not exhibit a consistent trend; however, it was observed that changes in the TDS caused signiยฎcant changes in the activity of chloride ions in solution. These ยฎndings are of interest in using CDs to formulate parenteral and ophthalmic solutions where the OP of the solutions must be considered.