RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism, initiated by the presence or introduction of double-stranded RNA, known as small interfering RNA (siRNA). The siRNA can serve as a guide for enzymatic and sequence-specific degradation of mRNAs, probably by associating with a multi-component nuclease. We have recently discovered and cloned a novel inhibitory isoform of vascular endothelial growth factor (VEGF), VEGF_165b (Bates et al. 2002). In the absence of a VEGF_165b-specific antibody, we investigated if RNAi can be used to reduce production of this new isoform. A VEGF_165b-specific sequence of 21 nucleotides located across exon 7 and exon 9 of VEGF gene plus an additional 17 nucleotides of universal T7 promoter sequence and a complementary T7 promoter primer were synthesized separately, annealed at 95 °C for 5 min, and allowed to cool down to room temperature to obtain DNA template. Both sense and anti-sense template were constructed in the same way. The template was incubated in 50 µl reaction volume with T7 RNA polymerase at 37 °C for 2 h, then RNase-free DNase was added to remove the DNA template. The crude reaction cocktails of both sense and anti-sense were mixed and heated at 95 °C for 5 min, then slowly cooled down by incubation at 37 °C for 1 h to obtain double-stranded RNA. A different double-stranded RNA with a two nucleotides mismatch from the VEGF_165b-specific siRNA was also designed and produced as above (mismatch VEGF_165b siRNA). Both purified double-stranded RNAs (phenol : chloroform extraction) were transfected with pcDNA3-VEGF_165b vector into HEK 293 cells using LIPOfectamine PLUS; pcDNA-VEGF_165b vector alone was transfected into cells as control. The VEGF concentration in cell culture media was determined by ELISA 2 days after the transfection. VEGF concentration (mean ± S.E.M.) was reduced 8.32 ± 3.41-fold by co-transfection of VEGF_165b-specific siRNA compared with pcDNA3-VEGF_165b vector transfection alone. Co-transfection of mismatch VEGF_165b siRNA decreased VEGF concentration 1.91-fold (P < 0.05, ANOVA, compared with VEGF_165b siRNA). We have demonstrated that VEGF_165b siRNA can be used to specifically reduce expression of VEGF_165b protein. We suggest that siRNA transfection is a useful method to study the effect of reduced VEGF_165b production in vitro.