Transient receptor potential melastatin 8 (TRPM8) channels are known to be present in rat and human bladder tissue (Stein et al., 2004). Recent studies in our lab (Johnson et al., 2009) have shown that this channel is present and functional in rat vascular smooth muscle. Therefore, this project aimed to examine the functional expression of TRPM8 channels in the bladder of normal rats using isometric contractile studies along with immunohistochemistry. Bladders were taken from sacrificed male Sprague-Dawley rats (250-350g). Tissues were processed for histology and immunohistochemical staining. Bladder tissues labeled with anti-TRPM8 (N=4) showed positive staining in the urothelium, nerves and in cells with the morphological features of Interstitial Cells (IC) in the lamina propria and detrusor layers. Detrusor smooth muscle was not immunopositive for TRPM8 in contrast to DRGs which were used as positive controls. Isometric contraction studies were conducted in bladder strips pre-tensed to 1g with urothelium in tact or removed. TRPM8 agonist, menthol, (300μM in ethanol) failed to cause any contraction (N=4, n=16 where N=animals, n= preparations). Contraction (1.97±0.15 g; ave.±s.e, N=10, n=18) induced by carbachol (10 μM) was markedly relaxed on addition of menthol (72±5%, N=9, n=18, accounting for relaxation due ethanol alone; P<0.001, ANOVA, Tukey’s post hoc test). However, when protocols were repeated in the presence of the L-type calcium channel blocker, nifedipine (10 μM) menthol-induced relaxation was vastly reduced (93 ±4 %, N=9, n=14, not significant). Results for these protocols were unaffected by removal of urothelium. Both WS-12 (10 μM, N=6, n=8) and icilin (10 μM in DMSO, N=3, n=12) failed to have contractile or relaxatory effects with urothelium in tact. We conclude that in normal rat bladder, TRPM8 channels are not involved in contractile responses. This is consistent with the lack of TRPM8 staining in detrusor smooth muscle. The expression of TRPM8 in urothelium, nerves and IC may be related to sensory mechanisms in the bladder.