The natural vitamin, pyridoxine, in the millimolar range is toxic to cultured rat hepatoma cells. A pyridoxine-resistant Fu5-5 rat hepatoma cell line was established by a stepwise increase in the concentration of pyridoxine in the medium. The newly established cell line, referred to as clone 10 (Cl.10), is resistant to killing by pyridoxine in concentrations up to 5 mM. Saturation kinetics for the uptake of [3H]pyridoxine into Fu5-5 and Cl.10 cells revealed that Fu5-5 cells take up 10 times more [3H]pyridoxine than do Cl.10 cells. Whereas the Vmax value for the uptake of [3H]pyridoxine was the same for both cell lines, the apparent Km for the Cl.10 cells was 12.5 microM compared to 0.71 microM for the Fu5-5 cells. However, intracellular levels of pyridoxal 5'-phosphate were 69% higher in Cl.10 cells than in the parental line. The resistant line is neither a permeability mutant nor deficient in pyridoxal kinase. Cl.10 cells contain 37% more adenosine 5'-triphosphate than do Fu5-5 cells and have a mitochondrial volume that is 50% greater than that of the parental line. In the absence of pyridoxine in the medium, Cl.10 cells revert to parental type with respect to pyridoxine uptake but not with respect to resistance to killing. They also maintain an enlarged mitochondrial volume. Thus, increased mitochondrial volume may be related to the development of resistance to high levels of pyridoxine.