Purpose. A new mucus-secreting in vitro drug absorption model based on monolayers of goblet-cell like sub-clones of the human colon carcinoma cell line HT29 obtained by methotrexate (MTX) treatment was investigated. Methods. Twelve sub-clones were isolated and characterized by light microscopy (LM), transelectron microscopy (TEM), confocal laser scanning microscopy (CLSM), transepithelial electrical resistance (TEER) and the transport of a paracellular marker FITC-Dextran (Mw 4400) (FD-4). Results. Significant differences of microscopical appearance, TEER-values and permeability of FD-4 between the sub-clones were evident. However, two of them, namely MTX-DI and MTX-E12, formed tight confluent monolayers with a thick mucus-layer on the apical surface. They were used to compare the apparent permeability coefficient (P-app) of a series of lipophilic drugs, which should be affected by the mucus-layer, namely barbiturates (barbituric acid, barbital, phenobarbital, methylphenobarbital and heptabarbital) and testosterone, as a reference, to mucus-free Caco-2 cells. The permeability of drugs with a partition coefficient (log P) > I was decreased in the mucus-producing cell lines. Testosterone, the most lipophilic compound, showed a decrease of up to 43%. Conclusions. We demonstrated that the mucus layer is a significant barrier to drug absorption for lipophilic drugs. In conclusion, our model may serve as a suitable in-vitro cell culture model to study the influence of the mucus layer on drug diffusion.