Selection of optimal antisense constructs is still a problem. Among a huge number of antisense oligonucleotides (AS-ONs) only a small piece show inhibitory efficacy. We want to develop an enhanced strategy for specific selection of effective AS-ONs based on prediction of secondary structure of the target messenger RNA (mRNA) and analysis of thermodynamic properties of the mRNA/AS-ON hybrid. Numerous AS-ONs targeted on human tissue factor (TF) mRNA were investigated to evaluate the relevance of different thermodynamic and structural properties on inhibitory efficacy. Cell viability, TF protein and TF mRNA were determined after transfection of bladder cancer cell line J82. Inhibitory efficacy was related to GC content, target region within the TF mRNA and stability of the mRNA/AS-ON hybrid or affinity of the AS-ON to the target mRNA. We found effective AS-ONs targeted on translated region or 3'-untranslated region of TF RNA. We also detected a great correlation between inhibitory efficacy and GC content as well as stability of the mRNA/AS-ON hybrid.