VEGF₁₆₅b is an alternatively spliced isoform of VEGF that has been shown to inhibit endothelial cell proliferation, migration and vasodilatation induced by VEGF₁₆₅. It is down-regulated in renal cell carcinoma and it has been hypothesised that it is an endogenous anti-angiogenic isoform of VEGF (Bates et al. 2002). To determine whether VEGF₁₆₅b was also expressed in prostate, and whether it was down-regulated in prostate cancer, we measured expression of VEGF₁₆₅b mRNA in prostate using fresh transurethral resection (TUR) chips and archival formalin-fixed paraffin-embedded (FFPE) tissue. TUR chips and archival tissue were obtained from patients who had undergone TUR of the prostate for bladder outlet obstruction and radical prostatectomy for localised prostate cancer, respectively. All tissues were surplus to requirements for histology, and were collected with Ethics Committee approval. Total ribonucleic acid (RNA) was isolated from fresh tissue based on the guanidium thiocynate phenol-chloroform extraction method described by Chomczynski & Saachi (1989). RNA was recovered from FFPE tissue using proteinase K digestion followed by phenol-chloroform extraction (Krafft, 1997). Nineteen prostate carcinoma tissue (9 TUR and 10 FFPE) and 27 benign prostate (17 TUR and 10 FFPE) were analysed. Paired malignant and benign portions were obtained from each FFPE specimen. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using intron spanning primers designed to amplify only those samples containing the novel exon of VEGF₁₆₅b, exon 9. PCR conditions were optimised and products were analysed by agarose gel electrophoresis. Bands corresponding to the amplification of VEGF₁₆₅b were detected in significantly more (82 %) benign TUR chips than the malignant ones (11 %, P < 0.05, Fisher’s exact test). VEGF₁₆₅ on the other hand was expressed in both normal and malignant tissue. VEGF₁₆₅b was detected in all the FFPE tissues but the mean intensity of the bands from the benign part of the prostate was significantly higher compared with that where prostatic intra-epithelial neoplasia (PIN) was found. VEGF₁₆₅b, a novel VEGF isoform that is potentially anti-angiogenic, can be detected in fresh and even archival prostate tissue. It appears to be down-regulated in carcinoma of the prostate, indicating both transcriptional and splicing control of VEGF is involved in the angiogenic switch in prostate cancer.