It is widely accepted that ATP secretion in diverse cells involves pannexin 1 (Panx1) that functions as an ATP-permeable channel. We analyzed the permeability of Panx1 channels heterologically expressed in HEK-293 cells to a variety of anions, including ATP. As was demonstrated in electrophysiological experiments, relative permeabilities of studied species follow the sequence: C1- > MeSO4 > gluconate >> HEPES. This sequence suggests that ATP, which is more than twice as large as HEPES by mass, is most likely to be negligibly permeable to Panx1 channels. This inference was verified in experiments, where ATP secretion from Panx1-positive HEK-293 cells was assayed with the ATP-biosensor approach. It was shown that the heterologous expression of Panx1 in HEK-293, which normally are not ATP-secretive, did not endow transfected cells with the ability to liberate ATP in response to stimulation. Our data indicate that Panx1 alone forms anion channels with too low ATP permeability to mediate the secretory function. Nevertheless, the possibility still remains that certain ATP-permeable channels are heteromers of Panx1 and some other channel subunit(s).