BACKGROUND: Acyclovir has pharmacokinetic limitations, including poor oral bioavailability of 15%-30%, high variability, and short elimination half-life of 2.3 hours. These limitations necessitate frequent administration of acyclovir, up to five times daily, leading to poor patient compliance, which in turn leads to a reduction in therapeutic efficacy and development of resistance. METHODS: A gastroretentive sustained-release (GR) formulation of acyclovir, based on a combination of swelling and mucoadhesive mechanisms, has been developed. Composition has been optimized after evaluation of different polymers, carbomer, polyethylene oxide, and sodium alginate alone and/or in combination. GR formulations were characterized for in-process quality-control tests, drug release and release rate kinetics, similarity factor analysis, swelling index, and matrix erosion. RESULTS: A formulation containing a combination of carbomer and polyethylene oxide had the highest similarity of drug release compared with a target drug-release profile obtained by pharmacokinetic simulations. The measurement of mucoadhesive strength, carried out with a texture analyzer, showed that the mucoadhesive strength of the GR formulation was significantly higher than that of the immediate-release (IR) tablet. The optimized GR formulation was found to be retained in the upper part of the gastrointestinal tract for 480 minutes; the IR tablet was retained for only 90 minutes as measured using a gastrointestinal retention study in albino rabbits. The GR formulation was also found to maintain more sustained plasma concentrations than the IR tablet. Mean residence time of the GR formulation was 7 hours versus 3.3 hours for the IR formulation. The relative bioavailability of the GR formulation was 261% of the IR formulation. CONCLUSION: The GR formulation of acyclovir, based on swelling and mucoadhesive mechanisms, has prolonged retention in the upper gastrointestinal tract, sustained in vitro drug release, prolonged in vivo absorption, and better bioavailability than the IR formulation. Such a formulation would improve patient compliance and increase the efficacy of therapy.