We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb 3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin–streptavidin and the digoxigenin–anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb 3+ –EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin–streptavidin and detection via digoxigenin–anti-digoxigenin offer 2–3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.