ATP is an electrically charged molecule required to obtain the energy necessary for cellular activity; in addition, it is a signalling molecule. Usually, the controlled secretion of ATP occurs through the exocytosis of granules and vesicles. However, in some cells, and under certain circumstances, other mechanisms control ATP release. It has been suggested that connexins may be an alternative pathway for ATP release. In gap junctions, connexins link the cytoplasm of two adjacent cells by establishing an intercellular channel and control the passage of ions and molecules up to 1 kDa. The channel is formed by two moieties called hemichannels, or connexins. We have investigated the release of ATP from Xenopus oocytes through hemichannels formed by connexin 38 (Cx38), an endogenous, specific type of connexin. Calcium-free solution reversibly activates an inward current (830 ± 202 nA, p<0.05) that is inhibited by 1.5 mM octanol (88% inhibition) and 50 μM flufenamic acid (76% inhibition). This calcium-sensitive current depends on Cx38 expression: it is decreased (268 ± 64 nA, p<0.05) in oocytes injected with an antisense oligonucleotide against Cx38 mRNA (ASCx38) and is increased in oocytes overexpressing Cx38 (1767 ± 85 nA, p<0.001). The opening of Cx38 hemichannels also permeates for small molecules (<1 KDa) like Lucifer Yellow (LY) demonstrating that it is involved in the generation of the inward current generated in the absence of free calcium ions. The activation of the endogenous connexons by calcium-free solution also induced the release of ATP (0.04 ± 0.023 pmol ATP oocyte^-1 min^-1, n = 6), which was inhibited by 50 μM flufenamic acid (61% inhibition), 1.5 mM octanol (66% inhibition) and ASCx38 (72% inhibition) and increased by Cx38 overexpression (300% increase). Altogether, these results strongly suggest that the activation of Cx38 hemichannels is responsible for the release of ATP.