TRPM3 is a TRPM subfamily member reported to be activated by reduced extracellular osmolarity [1] and sphingolipids closely related to, and including, D-erythro-sphingosine [2]. While conventional TRP channel inhibitors such as lanthanides and 2-APB have been used to characterise TRPM3, these agents are non-specific. Using previously described methods to target antibodies to the third extracellular loop in the channel structure [3] we developed the subtype specific antibody TM3E3. Targeting of the antibody near to the channel pore allows for inhibition of TRPM3 channel activity. Preincubation with TM3E3 produced about a 40% block of calcium influx through the TRPM3 channel, as measured by 96-well plate calcium fluorimetry, and direct application of TM3E3 during patch-clamp recording. Preadsorption of TM3E3 to its antigenic peptide prevented its blocking effect. TM3E3 had no effect on the closely related channel TRPM2, or other TRP family members TRPC5 and TRPV4, confirming its specificity for TRPM3. Recently, novel activators for TRPM3 have been suggested. In conjunction with TM3E3, these modulators have been used to study native TRPM3 in human saphenous vein smooth muscle cells (HSV cells). Messenger RNA encoding TRPM3 was detected by RT-PCR and peptide-specific labelling of the cells occurred with TM3E3. Sphingosine and its analogues evoked calcium responses in HSV cells that could be inhibited by gadolinium or 2-APB, and sphingosine-evoked calcium entry was partially suppressed following preincubation with TM3E3. This inhibitory effect was mimicked by siRNA that suppressed TRPM3 gene expression. The data show a novel TRP channel tool for studies of endogenous channels and suggest TRPM3 is a functional calcium-entry channel of vascular smooth muscle cells.