Anoctamin-1 (ANO1) is a Ca²⁺-activated Cl^- channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of the conductance. Several new antagonists of ANO1 with relatively high affinity and selectivity have been suggested to have usefulness for experimental and potentially for therapeutic purposes. We investigated the effects of intracellular Ca²⁺ (100-1000 nM), a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on the block of ANO1 channels expressed in HEK 293 cells. Tests were performed on a variety of naturally occurring splice variants of ANO1 using whole cell and excised patch configurations of the patch clamp technique. Block of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent upon [Ca²⁺]_i. Increasing [Ca²⁺]_i reduced the potency of this blocker. Similar Ca²⁺-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca²⁺ might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca²⁺]_i depended upon the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends upon [Ca²⁺]_i, suggesting a need for caution when using ANO1 blockers to determine the role of ANO1 in physiological functions and in use as therapeutic agents.