The photophysical and photobiological properties of a new class of cyclometalated ruthenium(II) compounds incorporating it-extended benzo[h]imidazo[4,5-f]quinoline (IBQ) cyclometalating ligands (CAN) bearing thienyl rings (n = 14, compounds 1-4) were investigated. Their octanol water partition coefficients (log P-o/w) were positive and increased with n. Their absorption and emission energies were red-shifted substantially compared to the analogous Ru(II) diimine (NAN) complexes. They displayed CAN-based intraligand (IL) fluorescence and triplet excited-state absorption that shifted to longer wavelengths with increasing n and NAN-based metal-to-ligand charge transfer (MLCT) phosphorescence that was independent of n. Their photoluminescence lifetimes (tau,m) ranged from 4-10 ns for (IL)-I-1 states and 12-18 ns for (MLCT)-M-3 states. Transient absorption lifetimes (zTA) were 5-8 mu s with 355 nm excitation, ascribed to (IL)-I-3 states that became inaccessible for 1-3 with 532 nm excitation (1-3, tau(TA) = 16-17 ns); the 3IL of 4 only was accessible by lower energy excitation, tau(TA) = 3.8,us. Complex 4 was nontoxic (EC50 > 300 mu M) to SK-MEL-28 melanoma cells and CCD1064-Sk normal skin fibroblasts in the dark, while 3 was selectively cytotoxic to melanoma (EC50=5.1 mu M) only. Compounds 1 and 2 were selective for melanoma cells in the dark, with submicromolar potencies (EC50 = 350-500 nM) and selectivity factors (SFs) around 50. The photocytotoxicities of compounds 1-4 toward melanoma cells were similar, but only compounds 3 and 4 displayed significant phototherapeutic indices (PIs; 3, 43; 4, >1100). The larger cytotoxicities for compounds 1 and 2 were attributed to increased cellular uptake and nuclear accumulation, and possibly related to the DNA-aggregating properties of all four compounds as demonstrated by cell-free gel mobility-shift assays. Together, these results demonstrate a new class of thiophene-containing Ru(II) cyclometalated compounds that contain both highly selective chemotherapeutic agents and extremely potent photocytotoxic agents.