Corticosteroids and macrolide immunomodulators such as tacrolimus are effective drugs for the topical treatment of inflammatory eye diseases like allergic conjunctivitis or dry eye. However, tacrolimus is practically insoluble in aqueous solutions and is therefore currently formulated as dispersion. This leads to low bioavailability. Here, we present a novel pharmacologically acceptable, aqueous formulation of tacrolimus based on the "Marinosolv formulation platform". Marinosolv allows the solubilization and thereby improvement of the bioavailability of many otherwise practically insoluble drugs, since dissolved drugs permeate faster into tissues, including ocular tissues. To visualize the benefits of Marinosolv in ophthalmic formulations, we investigated the permeation of a fluorescently labeled estradiol dissolved in Marinosolv compared to a formulation containing the compound as dispersion. Permeation was studied ex-vivo and in-vivo in porcine eyes. Further, we evaluated the improved permeation of topically applied tacrolimus dissolved in Marinosolv compared to a commercially available topically applied tacrolimus dispersion. The Marinosolv formulation was also compared to oral tacrolimus treatment, the standard application route for this drug in case of severe posterior uveitis. Finally, the ocular tissue levels of tacrolimus in all groups were determined using HPLC/MS. We demonstrated that tacrolimus dissolved in Marinosolv reached significantly higher levels in ocular tissues compared to the marketed topical product or after oral application and thus may be a suitable novel option for the treatment of several eye diseases, such as allergic conjunctivitis or uveitis. Thus, Marinosolv may be considered as a new vehicle for tacrolimus eye drops.