AbstractPeptide–oligonucleotide conjugates have frequently been synthesized to improve cellular delivery of antisense or antigene compounds, to allow the immobilization of peptide and protein conjugates on DNA arrays, or to decorate nucleic acid architectures with peptide functions. In such applications, the site of conjugation is of little importance, and peptides have predominantly been appended to one of the terminal ends of the oligonucleotide by using an oxime‐, thioether‐, or disulfide‐linkage or native chemical ligation. We, herein, demonstrate the first coupling of peptides to sequence internal sites. This attachment mode provides better control of the spatial arrangement of peptides presented by self‐assembled nucleic acid scaffolds. Internal modification requires special phosphoramidite building blocks that can be used in automated DNA synthesis. For this purpose, Fmoc/StBu‐protected cysteine was attached via an aminopropargyl linker to the C5‐position of uridine. The rigid triple bond conferred a high reactivity in native chemical ligation reactions of 5–6mer peptide thioesters with up to 15 nucleotides long oligonucleotides. The desired peptide–oligonucleotide conjugates were obtained in high yields after purification. UV melt experiments revealed that the peptide modification does not hamper nucleic acid hybridization. This finding marked an important step in our research program devoted to studies of multivalent presentation of peptides via modular assembly of nucleic acid complexes. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 397–404, 2010.