To study the mechanism of amide formation between carboxylic acid and amine in aqueous media using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), hydrogels with two different types of carboxyl group locations were employed as substrates containing the carboxylic acid, while ethylenediamine and benzylamine were used as amine. In parallel, a study was undertaken with cyclizable carboxylic acids (maleic acid and poly(acrylic acid) and noncyclizable carboxylic acids (fumaric acid and poly(ethylene glycol) with the terminal carboxyl groups) to assess the reaction products by 13C-NMR and IR. EDC rapidly lost its activity in aqueous media of low pH, producing the corresponding urea derivative, but was very stable at neutral and higher pH regions. EDC could react with carboxyl groups at a relatively narrow low pH range such as 3.5-4.5. If carboxyl groups were cyclizable, they would react quickly with EDC producing carboxylic anhydrides, which formed the corresponding amides when amine compounds were present. On the other hand, a trace of amide was formed in the case of noncyclizable carboxylic acids. In addition, an excess of EDC caused an undesired side reaction to form stable N-acylurea, regardless of the special location of carboxylic acids.