Five boronated DNA-intercalating compounds [5-ortho-carboranyl phenanthridinium (5-o-CP), 5-para-carboranyl phenanthridinium (5-p-CP), 6-para-carboranyl phenanthridinium, water-soluble boronated phenanthridinium and water-soluble boronated acridine (WSA1)], primarily developed for boron neutron capture therapy (BNCT), were analysed regarding their binding in cultured human malignant glioma spheroids. Comparisons were made with the corresponding DNA intercalators ethidium bromide and acridine orange. Octanol/phosphate buffered saline-water coefficients were determined for all compounds, and it was found that the most lipophilic (5-o-CP and 5-p-CP) were most toxic and accumulated high amounts of boron in monolayer cells. These compounds bound primarily in the outermost part of spheroids with poor penetration into the inner region, even after 2 days of continuous exposure. On the other hand, the most hydrophilic compound (WSA1) showed lower toxicity and lower boron accumulation in monolayer cells, and rapid binding in the inner region of spheroids. A reasonable explanation for this observation is that the lipophilic compounds interact mainly with lipophilic parts of the cells, like cellular membranes, and therefore rapidly binds to cells, preventing penetration and binding to cells in the deeper region of the spheroids. The possibility of using these compounds for BNCT are discussed.