The aim of the present study was to characterize the vasorelaxant activity of a natural diterpene extracted from Croton zambesicus, ent-18-hydroxy-trachyloban-3-one (DT6), and of a synthetic diterpene of similar structure, ent-trachyloban-14,15-dione (DT10) in rat aorta. DT6 and DT10 inhibited aorta contraction in a concentration-dependent manner. DT10 was more potent than DT6 and both inhibited 100 mM KCl-induced contraction more potently than noradrenaline-evoked contraction. NOS inhibition did not significantly affect the effect of DT6 on KCl-evoked contraction whereas it significantly increased the inhibitory potency of DT10. In fura-2-loaded aorta rings stimulated with 100 mM KCl solution, DT10 simultaneously inhibited the contraction and the increase in cytosolic calcium. Furthermore, DT10 significantly inhibited calcium channel current recorded by the patch-clamp technique in human neuroblastoma SH-SY5Y cells. DT6 and DT10 as verapamil potentiated the relaxation evoked by Bromo-cGMP in noradrenaline-contracted aorta, in agreement with the known synergy of the actions of cGMP and Ca²⁺ channels blockers. However, despite the potentiation of the response to Bromo-cGMP, DT6, DT10 and verapamil depressed the relaxation evoked by endogenous and exogenous NO. In conclusion, these data suggest that the vasorelaxant activity of DT is associated with the blockade of the L-type voltage-operated calcium channels. Inhibition of NO-dependent relaxation could be related to a decrease in the availability of NO.