Long-range photoinduced electron transfer has been systematically examined in a series of small DNA duplexes covalently modified with ethidium and Rh(phi)(2)bpy(3+) through time-resolved and steady-state measurements of fluorescence quenching. Fast fluorescence quenching (k greater than or similar to 10(10) s(-1)) is observed for this donor/acceptor pair noncovalently bound to DNA, and transient absorption studies allow the assignment of this quenching to an electron-transfer mechanism. In the duplexes modified with tethered intercalators, intrahelix fluorescence quenching attributed to electron transfer occurs at distances up to 30 Angstrom. Over a donor/acceptor separation of similar to 20 Angstrom, approximately 30% of the ethidium fluorescence is quenched, while at a separation of similar to 30 Angstrom, approximately 10% quenching is observed. Time-resolved measurements indicate that this quenching is primarily static. Fluorescence polarization and melting studies indicate that the intercalators are rigidly bound, enhance helix stability, and the duplex populations are structurally homogeneous. The distance dependence of the quenching yield observed in these duplexes is shallow, but the quenching reaction is highly sensitive to stacking perturbations. Changes in the quenching yield with melting are directly correlated with hypochromicity associated with base stacking. Moreover, in duplexes containing a highly disruptive CA mismatch, large decreases in the quenching efficiency are observed, while with a well-stacked GA mismatch, electron transfer proceeds efficiently. Hence, in these covalently-modified assemblies, DNA-mediated electron transfer is found to depend only weakly on donor/acceptor separation, when compared to protein systems, but is highly sensitive to perturbations in base stacking.