The underlying matrix plays an important role in the adhesion, proliferation and differentiation processes of Caco-2 cells. When culturing these cells for pharmaceutical purposes it is essential to know the influence of different supports on morphological and functional cell parameters. The impact of polystyrene, Matrigel-coated polystyrene, glass and nanostructured Easy-To-Clean (ETC01) slides was investigated over time by real-time quantitative reverse transcription polymerase chain reaction, enzymatic assays and immunofluorescent staining techniques. Compared to polystyrene, ETC01 slides induced cellular activities towards functional differentiation after short cultivation times. Glass significantly accelerated the differentiation process up to day 10 in culture, while Matrigel-coating had no significant benefit. By day 21 postseeding, the phenotype had equalized as indicated by constant brush border enzyme activity and villin mRNA expression masking the initial differences between the supports. The accelerated differentiation on specific matrices could be advantageous as it may enable cultured monolayers to be used earlier, and has to be considered when interpreting and comparing results.