['Rosmarinus officinalis L. is a plant used around the world for its properties to cure pain in several conditions, such as arthritic and abdominal pain or as an antispasmodic; however, there are no scientific studies demonstrating its spasmolytic activity. Therefore, the aim of the present study was to investigate the effect of an ethanol extract from Rosmarinus officinalis aerial parts and the possible mechanism involved by using rings from the isolated guinea pig ileum (IGPI).', 'The IGPI rings were pre-contracted with potassium chloride (KCl; 60 mM), acetylcholine (ACh; 1 × 10(-9) to 1 × 10(-5)M) or electrical field stimulation (EFS; 0.3 Hz of frequency, 3.0 ms of duration and 14 V intensity) and tested in the presence of the Rosmarinus officinalis ethanol extract (150, 300, 600 and 1 200 μg/mL) or a referenced smooth muscle relaxant (papaverine, 30 μM). In addition, the possible mechanism of action was analyzed in the presence of hexametonium (a ganglionic blocker), indomethacine (an inhibitor of prostaglandins), l-NAME (a selective inhibitor of the nitric oxide synthase) and nifedipine (a calcium channel blocker).', 'Rosmarinus officinalis ethanol extract exhibited a significant and concentration-dependent spasmolytic activity on the contractions induced by KCl (CI(50) = 661.06 ± 155.91 μg/mL); ACh (CI(50) = 464.05 ± 16.85 μg/mL) and EFS (CI(50) = 513.72 ± 34.13 μg/mL). Spasmolytic response of Rosmarinus officinalis (600 μg/mL) was reverted in the presence of nifedipine 1 μM, but not in the presence of hexamethonium 0.5mM, indomethacine 1 μM or L-NAME 100 μM.'] Does spasmolytic activity of Rosmarinus officinalis L. involve calcium channels in the guinea pig ileum?