A gastric retention formulation (GRF) made of naturally occurring carbohydrate polymers and containing riboflavin was tested in vitro for swelling and dissolution characteristics as well as in fasting dogs for gastric retention. The bioavailability of riboflavin, a drug with a limited absorption site in the upper small intestine, from the GRF was studied in fasted healthy humans and compared to an immediate release formulation. It was found that when the GRF is dried and immersed in gastric juice it swells rapidly and releases its drug content in a zero-order fashion for a period of 24 h. In vivo studies in dogs showed that a rectangular shaped GRF stayed in the stomach of fasted dogs for more than 9 h, then disintegrated and reached the colon in 24 h. Endoscopic studies in dogs showed that the GRF hydrates and swells back to about 75% of its original size in 30 min. These in vivo results correlated with in vitro results. Pharmacokinetic parameters determined from urinary excretion data from six human subjects under fasting conditions showed that bioavailability depended on the size of the GRF. The biostudy indicated that bioavailability of riboflavin from a large size GRF was more than triple that measured after administration of an immediate release formulation. Deconvolved input functions from biostudy data suggest that the large size GRF stayed in the stomach for about 15 h.