Ditercalinium (DIT; NSC 335153), a 7H-pyridocarbazole dimer, was reported to be capable of binding with high affinity to DNA by bisintercalation. Both the cytostatic and cytotoxic effects of this drug have been attributed to its binding to DNA. DIT inhibits the growth and is cytotoxic to Friend erythroleukemia (FL) cells. When FL cells were treated with 0.5-2.5 microM DIT and then stained with acridine orange (AO), which differentially stains DNA and RNA, the green, orthochromatic fluorescence representing AO binding to DNA was unchanged, while the metachromatic red luminescence characteristic of AO binding to RNA was reduced by as much as 40% in 4 hr; the effect was DIT-concentration dependent. The reduction in RNA stainability by DIT in the absence of any significant decrease in RNA content, was also observed with another RNA-specific fluorochrome, pyronin Y (PY). These results indicate that in live cells DIT preferentially binds to RNA rather than DNA, preventing stainability of the former by the monointercalating dyes AO and PY. When FL cells were exposed to 10 microM DIT after being first permeabilized by ethanol, the subsequent stainability of DNA in these cells was reduced by up to 67% and RNA by up to 44%, indicating that under these conditions DIT binds to both DNA and RNA. This observation was confirmed by competition experiments between AO and DIT bound to DNA or RNA in permeabilized cells mixed with equivalent numbers of RNA-containing (DNase-treated) or DNA-containing (RNase-treated) cells, respectively. The mechanisms that protect DNA against binding by DIT in live cells are unknown but are lost in fixed cells and may be related to maintenance of cellular and/or nuclear membrane integrity. If the propensity for other intercalating drugs to bind to RNA in live cells is correlated with their antitumor activity as is DIT, the rationale for designing new drugs based solely on their affinity for DNA should be reevaluated.