The aim of this study is to determine the effective concentrations of chlorhexidine on the release for prolonged periods of time from a novel hydrogel system. A hydrogel that exhibits a volume phase transition in response to temperature was synthesized by radiation copolymerization of ethylene glycol vinyl ether and butyl vinyl ether in the presence of crosslinking agent, diethylene glycol divinyl ether. Hydrogel samples in the disc form (diameter, 10 mm and height, 1.5 mm) were utilized as a matrix for the release of an antimicrobial agent, chlorhexidine diacetate. Chlorhexidine loading into the hydrogel was performed by water sorption at 4°C, which allows high swelling and thus high loading capacity, i.e., ∼36 mg drug per gram of dry gel. Chlorhexidine release was examined as short-term (24 h) and long-term (27 days) by UV spectrophotometer. Microbial studies were carried out by micro-dilution method in order to determine the effectiveness of the drug release. Minimum inhibitory concentration values for the pathogens of Streptococcus mutans and Lactobacillus casei were determined. The long-term chlorhexidine release is initially very fast. After that, the drug release reaches a slow but a steady rate. Such a release pattern provides an effective drug release. The prolonged release of chlorhexidine is continued up to the 27th day. MIC values for the two pathogens have been shown that the release rate from disc is effective to inhibit the growth of pathogens. These in vitro drug release results suggested that the thermosensitive hydrogelic system developed in this study can be evaluated as a delivery system for the release of chlorhexidine. © 2007 Wiley Periodicals, Inc.