Chitosan is currently one of the most promising polymers in development of new drug delivery systems due to its biodegradability, biocompatibility, good mucoadhesive properties and positive biological effects. The aim of this study was to prepare chitosan hydrogel suitable for incorporation of liposomes with dilti-azem chloride, and to evaluate release of the drug from liposome-in-gel formulation destined for topical anal fissure therapy. Because of its superior wound healing properties, high molecular weight chitosan was chosen to prepare 2.2% (w/w) hydrogel. Conventional and propylene glycol liposomes with diltiazem chlo-ride were incorporated into the chitosan gel and marketed hydroxyethyl cellulose gel as well. Both types of liposomes were smaller than 200 nm, while conventional liposomes were able to incorporate higher amount of the drug. In vitro release studies of diltiazem chloride from liposomes-in-hydrogel formulation were performed using so-called agarose method during 24 hours, in phosphate buffer pH 6.8. Incorporation of liposomes into the both types of hydrogel prolonged release of the drug in comparison to control hydrogels. The results showed that type of both liposomes and hydrogel affected the rate and amount of the released drug. Diltiazem chloride release from the conventional liposomes was less than from elastic propylene gly-col liposomes, regardless of the type of hydrogel vehicle used. Both type of liposomes sustained the drug release, particularly when incorporated into the chitosan hydrogel. Administration of diltiazem chloride liposomes-in-chitosan hydrogel could provide sufficient retention time of diltiazem chloride at the site of application and additionally improve local therapy of anal fissure as a result of chitosan favorable wound healing properties.