We have studied parameters affecting DNA hybridization and lanthanide chelate complementation based signal formation in a separation-free solid-phase assay. This binary probe assay system consists of two probes labeled either with a europium carrier chelate or a light harvesting antenna ligand. One probe was immobilized on the microtiter well bottom in spot format while the other probe was free in solution. The probe concentration used in spotting, spacer length, and the choice and orientation of the either 3´ or 5´-end immobilized probe had significant impact on signal-to-background (S/B) ratios. The highest ratio was achieved by saturating the spot with the 5´-end immobilized antenna ligand probe separated from the solid support with a 25 nucleotide poly dT spacer. The obtained detection limit of 18 pM for synthetic Pseudomonas aeruginosa heat shock protein groES gene sequence was close to a 20-fold improvement compared to the previous assay. The dynamic range of the assay was three orders of magnitude.