Examination was made of the effect of annexin V on Ca²⁺ movement into large unilamellar vesicles (LUV) using fura-2, a calcium-sensitive fluorescent dye. To avoid the possible difficulties relating to the addition of annexin V and/or Ca²⁺ in fura-2-loaded LUV, the burst method was used. LUV, preincubated with rat annexin V in the presence of Ca²⁺, were collected by centrifugation and resuspended, and then burst with Triton X-100 in the presence of fura-2. Inward Ca²⁺ movement across the artificial lipid membrane was measured by determination of fura-2 fluorescence due to the leaked Ca²⁺ from ruptured LUV. The observed Ca²⁺ signal increased dependent on annexin V and Ca²⁺ concentrations, whereas bovine serum albumin did not affect this signal up to 1 μM. Thus, annexin V shows Ca²⁺ channel activity in LUV. K201, a novel 1,4-benzothiazepine, inhibited inward Ca²⁺ movement into LUV caused by annexin V in a dose-dependent manner. In the presence of 50 nM annexin V and 400 μM Ca²⁺, 3 μM K201 showed significant inhibition of Ca²⁺ movement due to annexin V, and 50% inhibition was achieved at 25μM K201. On the other hand, diltiazem had no such effect even at 30 μM. K201 is thus shown to have inhibitory activity on inward Ca²⁺ movement due to annexin V in artificial vesicles and may prove useful as a probe for elucidating the functions of annexin V in vivo.