One of the central molecules in capillary formation during angiogenesis is the integrin alphaVbeta3. The aim of this study was to inhibit alphaV-mediated angiogenesis in vitro using RNAs (siRNA) as well as antisense oligodeoxyribonucleotides (asON). Five siRNAs, against the alphaV chain of alphaVbeta3, and three asON, which had the respective sequence of the antisense sequence of three of the siRNAs molecules, were examined. Two of the siRNAs and their respective asON were designed on the basis of computer-predicted secondary structure analysis of alphaV mRNA. The different molecules were transfected into human umbilical vein endothelials cells (HUVEC) using lipofection. Following stimulation by PMA, two siRNAs showed a dose-dependent inhibition of PMA-induced alphaV mRNA and protein upregulation, as assessed by real-time RT-PCR and flow cytometry. At a concentration of 25 nM a complete inhibition of protein upregulation was found using siRNAs while transfection of the respective asON sequences reduced the protein upregulation only by 44%. To evaluate the anti-angiogenic potential a cell culture model of human angiogenesis based on the co-cultivation of endothelial cells and dermal fibroblasts was used. Transfection of the siRNA sequence (50 nM) resulted in an inhibition of the total length of capillary-like tubules by 40.6% in comparison to 21.1% using the respective asON sequence. In conclusion, siRNA-based downregulation of alphaV expression showed a stronger inhibition of capillary tube formation in an angiogenesis in vitro assay, than asON having the same sequence as the antisense strand of the siRNAs. Therefore, siRNAs are useful tools for functional alphaV knock-down experiments and might be a therapeutic alternative for antagonists which bind directly to the integrins alphaVbeta3 or alphaVbeta5.