This paper describes the design of a series of luminescent rhenium(I) polypyridine biotin complexes containing different spacer-arms, [Re(N-N)(CO)(3)(py-4-CH2-NH-biotin)](PF6) (py-4-CH2-NH-biotin = 4-(biotinamidomethyl)pyridine; N-N = 1,10-phenanthroline, phen (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline, Me-4-phen (2a), 2,9-dimethyl4,7-diphenyl-1,10-phenanthroline, Me-2-Ph-4-phen (3a), dipyrido[3,2-f.2',3'-h]quinoxaline, dpq (4a)), [Re(N-N)(CO)(3)(py-3-CO-NH-en-NH-biotin)](PF6) (py-3-CO-NH-en-NH-biotin = 3-(N-((2-biotinamido)ethyl)amido)pyridine; N-N = phen (1b), Me4-phen (2b), Me-2-Ph-2-phen (3b), dpq (4b)), and [Re(N-N)(CO)(3)(py-4-CH2-NH-cap-NH-biotin)](PF6) (py-4-CH2-NH-cap-NH-biotin = 4-(N- ((6-biotinamido) hexanoyl)ami nom ethyl)pyridine; N-N = phen (1c), Me4-phen (2c), Me-2-Ph-2-phen (3c), dpq (4c)). Upon irradiation, all of the rhenium(I)-biotin complexes exhibited intense and long-lived triplet metal-to-ligand charge-transfer (3 MLCT) (d pi,7(Re) -> pi*(diimine)) emission in fluid solutions at 298 K. The interactions of these biotin-containing complexes with avidin have been studied by 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assays, emission titrations, and competitive association and dissociation assays. On the basis of the results of these experiments, homogeneous assays for biotin and avidin have been designed.