Extracellular nucleotides signal via a large group of purinergic receptors. Although much is known about these receptors, the mechanism of nucleotide transport out of the cytoplasm is unknown. We developed a functional screen for ATP release to the extracellular space and identified Mcd4p, a 919-amino acid membrane protein with 14 putative transmembrane domains, as a participant in glucose-dependent ATP release from Saccharomyces cerevisiae. This release occurred through the vesicular trafficking pathway initiated by ATP uptake into the Golgi compartment. Both the compartmental uptake and the extracellular release of ATP were regulated by the activity of the vacuolar H+-ATPase. It is likely that the Mcd4p pathway is generally involved in non-mitochondrial ATP movement across membranes, it is essential for Golgi and endoplasmic reticulum function, and its occurrence led to the appearance of P2 purinergic receptors.