A group of compounds composed of pyridine, 2,2'-bipyridine and 2,2':6',2''-terpyridine as the energy absorbing, triplet sensitizing moieties and two or four carboxylic acids as the chelating groups were synthesized and their ability to enhance terbium ion luminescence was elucidated. The dicarboxylic acid derivatives form highly luminescent Tb3+ chelates with luminescence quantum yields approaching 50%. Additional groups, such as methoxy groups or additional pyridine rings in the aromatic structure can be utilized to shift the excitation maxima towards longer wavelengths in order to produce more practicable excitation for practical use. The ligands giving the highest Tb3+ emissions were further evaluated in a two-step fluorescence enhancement system constructed for specific binding assays based on the simultaneous use of three or four ions as the labels (Eu3+, Tb3+, Sm3+ and Dy3+). In the two-step procedure, the first step comprises dissociative fluorescence enhancement based on an acidic solution of an aromatic beta-diketone used as the luminogenic ligand, whereafter Eu3+ and Sm3+ are quantitated. In the second step, the lanthanide ions form new chelates with the added ligands. These ligands are able to transfer the excitation energy they have absorbed with high efficiency to the chelated ions and produce high ion luminescence for Tb3+ and Dy3+.