Gastrointestinal (GI) motility disorders such as irritable bowel syndrome (IBS) occur when coordinated smooth muscle contractile activity is disrupted, and little is known about the molecular mechanisms underlying GI tract relaxation. Here we utilise quantitative RT-PCR (qPCR) and organ bath pharmacology to study the contribution of various K+ channel subtypes to mouse intestinal muscle tension. Distal ileum and colon segments were dissected from adult C57BL/6 mice, suspended from a force transducer in an aerated organ bath containing Krebs solution at 32°C. Changes in longitudinal muscle tension were measured using LabChart® software (AD Instruments). Tissues were pre-contracted with 10 µM carbachol (CCh) or 60 mM KCl. Data are presented as mean ± SEM (n = 6, unless stated) and statistical analysis performed by t-test with significance at p < 0.05. qPCR analysis for 93 K+ channel α- and β-subunit genes was performed on independent ileum and colon sections from 3 animals. KCNJ8 (KIR 6.1) had the highest relative expression of the 15 KIR genes examined. 100 µM diazoxide had a small but significant relaxation of CCh contracted ileum (14.8 ± 2.8 %, p = 0.0034) and colon (15.2 ± 1.0 %, p = 0.0002). Of 40 KV genes analysed, KCNQ1 (KV 7.1) and KCND3 (KV4.3) showed the highest relative expression. 100 µM XE991 induced a small contractile response in ileum but 100 µM chromanol 293B was without significant effect (p = > 0.05). 100 µM retigabine induced a differential relaxation in CCh and KCl contracted ileum (54.3 ± 1.9 % vs 105.6 ± 2.3 %, p <0.0001) and colon (35.4 ± 3.1 % vs 100.8 ± 3.1 %, p <0.0001). 50 µM NS 6508 (KV4.3 activator) had limited relaxatory effect on CCh contracted ileum and colon (27.5 ± 3.7 % and 14.4 ± 3.5 %). Of 8 KCa genes analysed, KCNN4 (IKCa) and KCNMA1 (BKCa) showed the largest relative expression. 100 µM NS 19504 (BKCa activator) induced relaxation in both CCh contracted ileum (73.8 ± 4.6 %) and colon (87.3 ± 2.1 %). Across 14 K2P genes, KCNK1 (TWIK-1) and KCNK5 (TASK-2) showed the greatest relative expression in ileum and colon, with KCNK2 (TREK-1) prominent in ileum but not colon. K2P channel specific pharmacology is limited, but previous studies have described activators of mechano-gated K2P channels including TREK-1 (1 & 2). 100 µM riluzole induced a significant reduction in CCh contracted ileum (37.1 ± 3.9 %, p <0.0001) and colon (18.9 ± 4.4 %, p = 0.0023). Relaxation induced by 60 µM BL-1249 and the structurally related fluflenamic acid (100 µM) differed between ileum and colon; 93.1 ± 1.2 % vs 56.1 ± 4.2 % (p <0.001) and 63.3 ± 6.2 % vs 41.1 ± 3.4 % (p = 0.0108) respectively. These data reveal the heterogeneous expression of K+ channel subtypes along the mouse GI tract and highlight K+ channels as potential therapeutic targets for treating spasmodic gut disorders.