When the Caco-2 cell line is used as a model for transepithelial transport pathways, striking differences in transepithelial electrical resistance and in permeability of hydrophilic marker substances were reported. In this study, Caco-2 cell lines from different laboratories have been tested for permeability characteristics using hydrophilic and hydrophobic substances. To elucidate the vast differences in permeability, the following properties were monitored: growth characteristics, morphological homogeneity, metabolic activity, viability, and actin. Using lectin binding experiments and alkaline phosphatase staining, the degree of differentiation in Caco-2 clones was estimated. Our results are in agreement with observations made by others, demonstrating heterogeneity of the Caco-2 cell line. Caco-2 monolayers consist of several subpopulations. The variation in permeability characteristics of the Caco-2 cell line may be a result of a selection process promoted by the respective culture conditions yielding a varying composition of subpopulations.