Background and purpose:Isoform‐specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family.Experimental approach:A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole‐cell planar patch‐clamp and fura‐2 intracellular Ca2+ measurement.Key results:Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5–10 min. Ca2+ entry in TRPM3‐expressing cells was also partially inhibited by TM3E3 in a peptide‐specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response.Conclusions and implications:The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform‐specific ion channel blockers.British Journal of Pharmacology (2008) 155, 567–573; doi:10.1038/bjp.2008.283; published online 7 July 2008