The maxi-anion channel plays a classically recognized role in controlling the membrane potential through the chloride conductance. It also has novel functions as a regulated pathway for the release of the anionic signaling molecules ATP and excitatory amino acids from cells subjected to osmotic perturbation, ischemia, or hypoxia. Because hemichannels formed by pannexins and connexins have been reported to mediate ATP release from a number of cell types, these hemichannels may represent the molecular correlate of the maxi-anion channel. Here, we found that L929 fibrosarcoma cells express functional maxi-anion channels which mediate a major portion of swelling-induced ATP release, and that ATP released via maxi-anion channels facilitates the regulatory volume decrease after osmotic swelling. Also, it was found that the cells express the mRNA for pannexin 1, pannexin 2, and connexin 43. Hypotonicity-induced ATP release was partially suppressed not only by known blockers of the maxi-anion channel but also by several blockers of pannexins including the pannexin 1-specific blocking peptide (10)Panx1 and small interfering (si)RNA against pannexin 1 but not pannexin 2. The inhibitory effects of maxi-anion channel blockers and pannexin 1 antagonists were additive. In contrast, maxi-anion channel activity was not affected by pannexin 1 antagonists and siRNAs against pannexins 1 and 2. Although a connexin 43-specific blocking peptide, Gap27, slightly suppressed hypotonicity-induced ATP release, maxi-anion channel activity was not affected by Gap27 or connexin 43-specific siRNA. Thus, it is concluded that the maxi-anion channel is a molecular entity distinct from pannexin 1, pannexin 2, and connexin 43, and that the maxi-anion channel and the hemichannels constitute separate pathways for swelling-induced ATP release in L929 cells.