The gingiva is the target site for some topical drugs, but the permeability of human gingiva has not been systematically evaluated. Pigs are a common animal model for in vitro membrane transport studies. The objectives of this study were to: (a) determine the permeability coefficients of freshly excised human gingiva using model permeants, (b) compare the permeability coefficients of fresh human gingiva with those of fresh porcine gingiva, (c) evaluate the effect of freezing duration on the permeability of porcine gingiva, and (d) compare the permeability coefficients of fresh and cadaver (frozen) human gingiva. A goal was to examine the feasibility of using porcine gingiva as a surrogate for human gingiva. The potential of using frozen tissues in permeability studies of gingiva was also examined. Fresh and frozen porcine gingiva, fresh human gingiva, and frozen cadaver human gingiva were compared in the transport study with model polar and lipophilic permeants. The fresh porcine and human tissues showed similarities in the “permeability coefficient vs. octanol–water distribution coefficient” relationship. The porcine gingiva had a lower permeability than that of the human, with a moderate correlation between the permeability of the fresh porcine and fresh human tissues. The permeability of the porcine tissues for the model polar permeants increased significantly after the tissues were frozen in storage. Moreover, the frozen human cadaver tissue could not be utilized due to the high and indiscriminating permeability of the tissue for the permeants and large tissue sample-to-sample variabilities.