This in vitro study aims to develop a cell culture model that compares paracellular permeability (PP) with acute cytotoxicity (AC). Caco-2 cells were seeded in 96-well plates and on polycarbonate filter inserts. Confluent monolayers were exposed to increasing concentrations of 20 reference chemicals for 24-h and 72-h. Cytotoxicity was determined using MTT and NRU cell viability assays in 96-well plates. PP was measured using transepithelial electrical resistance (TEER) measurements, as well as passage of lucifer yellow (LY), [3H]-mannitol (both low mw indicators), and FITC-dextran (higher mw indicator) in culture inserts. Inhibitory concentrations 50% (IC50s) suggest that there were good correlations between 24-h and 72-h exposures. NRU IC50 values correlated better with TEER, which is consistent with the Registry of Cytotoxicity (RC; ICCVAM) database report. Both cell viability assays indicate that cytotoxicity occurs before TEER is compromised. In addition, 24-h and 72-h NRU assays, and 72-h TEER measurements, displayed the highest correlations with established rodent LD50s. PP experiments showed that passive paracellular transport of the tight junction markers, especially [3H]-mannitol, correlates with the IC50s determined with the viability assays and TEER measurements. Our AC/TEER/PP model thus allows for the differentiation between the concentrations necessary for AC and those needed to interfere with PP. We propose that the in vitro AC, TEER and PP results be used to compute a formula which can "normalize" and improve the predictive ability of in vitro acute cytotoxicity assays for in vivo lethality.