The purpose of this investigation was to study the influences of absorption enhancers in increasing oral bioavailability of Ganciclovir (GAN) by assessing the transepithelial permeation across cell monolayers in vitro and bioavailability in rats in vivo. The permeation of GAN across Caco-2 and MDCK cell monolayers in the absence/presence of dimethyl-b-cyclodextrin (DMbCD), chitosan hydrochloride (CH), sodium lauryl sulphate (SLS), and their combinations was studied for a 2-h period. GAN was administered to rats in absence/presence of absorption enhancers and drug contents in plasma were estimated. We found that the apparent permeability coefficient (Papp) of GAN in absence of absorption enhancers (control) were 0.261 AE 0.072 Γ 10 Γ6 and 0.486 AE 0.063 Γ 10 Γ6 cm/s in Caco-2 and MDCK cell monolayers, respectively, whereas in the presence of DMbCD, CH, SLS, and their combinations, Papp of GAN increased by 5-to 25-fold and 7-to 33-fold as compared to control in Caco-2 and MDCK cell monolayers, respectively. However, in rats, the maximum enhancement in bioavailability of GAN during coadministration of these absorption enhancers was only fivefold compared to GAN control. To conclude, the absorption enhancers-DMbCD, CH, SLS, and their combinations demonstrated significant improvement in transepithelial permeation and bioavailability of GAN.