Ethidium bromide (EB), an intercalating drug, has been shown to prevent the in vitro interaction of the estrogen receptor (R) with DNA (André et al., 1976). We have now studied the effect of this drug on the nuclear translocation of R in order to determine whether DNA integrity is needed for this translocation. In a cell-free reconstituted system made of purified nuclei and cytosol, the pretreatment of nuclei by EB prevented approximately half of the R nuclear translocation, but was unable to extract more than 17% of the E2-R previously translocated. A series of indirect evidences suggests that EB inhibits the nuclear translocation of R by interacting with nuclear DNA. The degree of the inhibition was related to the amount of drug bound to nuclei and was in agreement with the degree of ultrastructural modifications of chromatin. R was not irreversibly altered by the drug. The EB inhibition was only observed with DNA-containing particles and with estrogen receptor able to bind to DNA. In surviving uteri the drug also inhibited the R nuclear translocation. These resuts indicate two types of nuclear translocation of R, one sensitive and the other resistant to EB, and suggest that DNA is required for the EB-sensitive translocation.