A 3'-[[2-[N-(3-aminopropyl)-N-(2- hydroxyethyl)amino]ethylphosphoryl]oligodeoxyribonucleoside methylphosphonate 12-mer was synthesized using the Aha-CPG solid support [Thaden, J. and Miller, P.S. (1993) Bioconjugate Chem, companion paper in this issue]. The oligomer was conjugated at the 3' primary aliphatic amine with tetramethylrhodamine 5-isothiocyanate. The rhodamine linker/spacer was stable in 10% fetal calf serum. After enzymatic phosphorylation, the molecule was conjugated at the 5' phosphate with 4'-[N-(2-aminoethyl)aminomethyl]-4,5',8- trimethylpsoralen [(ae(AMT)]. The rhodamine/psoralen doubly-conjugated oligomer formed photoadducts with complementary single-stranded DNA and RNA oligonucleotides when irradiated with long-wavelength ultraviolet light. The efficiency of UV cross-linking slightly exceeded that of a colinear, psoralen-derivatized oligonucleoside methylphosphonate, and exhibited relationships with UV fluence and temperature that are characteristic for psoralen-conjugated methylphosphonates. The 1:1 complex formed with the oligodeoxyribonucleotide target could be detected by its red fluorescence. Mouse L949 cells grown in the presence of the double conjugate were shown by means of computer-assisted epifluorescence microscopy to have internalized it. There was an accumulation of intensely fluorescent points and spots in a juxtanuclear region of the cytoplasm, and a faint, diffuse signal in the entire cell area.