Considerable interest is focused on overcoming multidrug resistance (MDR) in cancer chemotherapy. The in vitro experiments to characterize P-glycoprotein's (P-gp) function and to decrease its effects have led to a variety of strategies such as addition of competitors or supplementation of the medium with oligonucleotides complementary to the 5'-end of the MDR1-mRNA. For the Caco-2 cell line, an in vitro model for absorption screening, expressing multiple transporters including P-gp, which pumps substances back into the apical solution, P-gp activity might mask other relevant transport proteins' activity. The objective of the present study was to construct a Caco-2 subline with reduced P-gp expression level. Caco-2 cells were transfected by electroporation with two different mammalian expression vectors, and the obtained subclones were investigated at RNA (Northern blotting, RT-PCR), protein (FACS analysis), and functional (transport studies) levels for reduction in P-gp expression. Northern blotting showed that the levels of transcription of the inserted gene were different among the several clones, but those results did not completely correlate with the FACS analysis for P-gp expression. The clones with the strongest reduction in P-gp expression detected by the FACS analysis also showed the lowest secretory fluxes of the P-gp substrate talinolol in transport studies. Repetition of FACS analysis after 7 and 24 months on 20 to 30 passage older subclones still showed reduction in P-gp expression and indicated that they are stably transfected. The new cell lines constructed in the present study provide the possibility to perform in vitro absorption studies in a cell system composed of differentiated enterocytes growing as a monolayer like the normal Caco-2 cell line but with a lower down to almost lacking expression of P-gp.