Abstract The influence of the molecular weight on mucoadhesive and penetration enhancement properties of three grades of hyaluronic acid (1878, 693 and 202 kDa) has been evaluated. The mucoadhesive properties were investigated using buccal and vaginal porcine mucosa by means of a tensile stress method and using rat jejunum by means of an inclined plane method. The mucoadhesive performances observed using animal tissues were compared with the mucoadhesive properties observed using submaxillary or gastric mucin dispersions. The penetration enhancement properties were investigated using porcine buccal epithelium membrane or vaginal tissue and a cell monolayer (Caco-2 cell line). Chitosan hydrochloride, already described as a penetration enhancer towards buccal and vaginal mucosae and Caco-2 cell monolayers, was used as reference. Aciclovir (acyclovir), a poorly soluble and absorbable drug, commonly used in the treatment of Herpes simplex virus (type I and II), was used as the model drug. Unlike chitosan hydrochloride, which does not show any mucoadhesive potential at pH close to neutrality (buccal and intestinal), all hyaluronic acid grades show mucoadhesive properties in all the environments considered (buccal, vaginal and intestinal). In all cases, a decrease in molecular weight of hyaluronic acid produced an increase in the mucoadhesive performance. The hyaluronic acid with the lowest molecular weight (202 kD) exhibited the best penetration enhancement properties, that, depending on the substrate under consideration, was either comparable with or even better than chitosan hydrochloride. Therefore, this grade would be the most promising for buccal, vaginal and intestinal delivery of aciclovir.