The photoreactivity of new RuII-oligonucleotide conjugates is investigated in the presence of their complementary strands. The goal is to determine the origins of different effects of parameters that control the photocrosslinking process of the two strands. Therefore, two RuII compounds, either [Ru(tap)3]2+or [Ru(tap)2phen]2+ (tap = 1,4,5,8-tetraazaphenanthrene, phen = 1,10-phenanthroline) with different oxidation powers, were tethered with different linkers to either the 5′- or 3′-phosphate end of the probe strand before hybridization with the complementary strand. These systems were studied by time-resolved emission spectroscopy, UV/Vis absorption experiments, PAGE and MS (ESI) analyses. The best yields of photocrosslinking (45 %) obtained with [Ru(tap)3]2+ tethered to the 3′-position are due to (i) a higher oxidation power of the complex and (ii) its attachment at the 3′-position. Indeed, this tethering favours the interaction of the Ru compound with the duplex and, therefore, inhibits its photodechelation. This work allows better design of sequence-specific DNA photodamaging agents prior to biological applications.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)