Glycine-Sepharose CL 6B, activated with 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), was used as a model compound to study the hydrolysis and aminolysis of immobilized NHS-activated groups. For comparison, the soluble analog N-t-BOC-glycine-NHS has been used. Coupling of amino compounds, such as aminoethanol, aminohexane, amino acids, and esters of amino acids, is fast and efficient in both organic medium and buffered aqueous medium, e.g., coupling of aminoethanol is complete within 1 min. Hydrolysis of the activated groups in aqueous medium is general base catalyzed and is, particularly in berate buffer and at higher pH (> 9.0), accelerated by addition of salt. The NHS-activated glycine-CL 6B is less sensitive toward hydrolysis as compared to N-t-BOC-glycine-NHS. When amino compounds are coupled to NHS-activated Sepharose in aqueous medium, the use of a low buffer concentration and a pH of 8.5-9.0, without salt, is recommended. In combination with salt, phosphate buffer is preferred.