In this study we investigated the mechanism underlying the spasmolytic action of ent-7α-acetoxytrachyloban-18-oic acid (trachylobane-360) and ent-7α-hydroxytrachyloban-18-oic acid (trachylobane-318), diterpenes obtained from Xylopia langsdorfiana, on guinea pig ileum. Both compounds inhibited histamine-induced cumulative contractions (slope = 3.5 ± 0.9 and 4.4 ± 0.7) that suggests a noncompetitive antagonism to histaminergic receptors. CaCl 2-induced contractions were nonparallelly and concentration-dependently reduced by both diterpenes, indicating blockade of calcium influx through voltage-dependent calcium channels (Ca v). The Ca v participation was confirmed since both trachylobanes equipotently relaxed ileum pre-contracted with S-(-)-Bay K8644 (EC 50 = 3.5 ± 0.7 × 10- 5 and 1.1 ± 0.2 × 10- 5 M) and KCl (EC 50 = 5.5 ± 0.3 × 10- 5 and 1.4 ± 0.2 × 10- 5 M). K + channels participation was confirmed since diterpene-induced relaxation curves were significantly shifted to right in the presence of 5 mM tetraethylammonium (TEA +) (EC 50 = 0.5 ± 0.04 × 10- 4 and 2.0 ± 0.5 × 10- 5 M). ATP-sensitive K + channel (K ATP), voltage activated K + channels (K V), small conductance calcium-activated K + channels (SK Ca) or big conductance calcium-activated K + channels (BK Ca) did not seem to participate of trachylobane-360 spasmolytic action. However trachylobane-318 modulated positively K ATP, K V and SK Ca (EC 50 = 1.1 ± 0.3 × 10- 5, 0.7 ± 0.2 × 10- 5 and 0.7 ± 0.2 × 10- 5 M), but not BK Ca. A fluorescence analysis technique confirmed the decrease of cytosolic calcium concentration ([Ca 2+] c) induced by both trachylobanes in ileal myocytes. In conclusion, trachylobane-360 and trachylobane-318 induced spasmolytic activity by K + channel positive modulation and Ca 2+ channel blockade, which results in [Ca 2+] c reduction at cellular level leading to smooth muscle relaxation. © 2011 Published by Elsevier B.V.