It is now well established that nicotine adversely affects the integrity of the blood-brain barrier (BBB). In contrast, nicotine has been reported to increase the transendothelial electrical resistance (TEER) of CaCo₂ colon cancer cells. In the present study, the effects of nicotine upon the TEER and sucrose permeability of ECV304/C6 co-cultures and, for comparative purposes, CaCo₂ cells has been investigated. Neither ECV304 nor C6 cells were found to express measurable membrane levels of nicotinic acetylcholine receptors, as assessed by [³H]-epibatidine binding. Nicotine treatment (0.01-1 μM) for up to 48 h had little or no effect upon the TEER or sucrose permeability of either ECV304/C6 co-cultures or CaCo₂ cells. It is concluded that in contrast to the situation for the BBB, ECV304 cells lack nicotinic acetylcholine receptors and the barrier properties of ECV304/C6 co-cultures are not affected to any important extent by nicotine. This study underlines the conclusions made by other authors that the ECV304/C6 co-culture system is of limited validity as a model of the BBB.