DHA and EPA, which are n-3 polyunsaturated fatty acids (PUFAs), has been reported to improve LGI disorders such as ulcerative colitis and Crohn's disease through their anti-inflammatory effects. However, there are few studies on the effects of these PUFAs on the motilities of LGI tracts such as ileum and colon, in which these inflammatory disorders frequently occur. In order to clarify the effects of DHA and EPA on LGI tract motilities, we examined their effects on the contractions of ileal and colonic longitudinal smooth muscles (SMs) isolated from the guinea pig, by comparing them with those of an n-6 PUFA, linoleic acid (LA). In both ileal and colonic SMs, DHA and EPA (3 × 10⁻⁵ M each) significantly inhibited any contractions due to acetylcholine (ACh), histamine, and prostaglandin (PG) F_2α, and these inhibitory effects were more potent than those of LA. DHA and EPA also significantly inhibited CaCl₂-induced contractions constructed in Ca²⁺-free high-KCl solution. Any contractions induced by ACh, histamine, and PGF_2α were completely suppressed by verapamil (10⁻⁵ M). These findings suggest that DHA and EPA could suppress LGI tracts' contractile functions excessively enhanced in association with inflammatory diseases partly through inhibition of voltage-gated Ca²⁺ channel-dependent ileal and colonic SM contractions.