Membrane trafficking is regulated, in part, by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family. ARF function depends on the controlled binding and hydrolysis of GTP. In vitro, the GTPase activity of yeast ARF proteins can be stimulated by Gcs1p. Although Gcs1p was implicated in the regulation of retrograde transport from the Golgi to the ER and in actin cytoskeletal organization, its intracellular functions and distribution remain to be established. Following subcellular fractionation of yeast grown in rich medium, Gcs1p was localized in denser fractions than it was in cells grown in minimal medium. In yeast grown in rich or minimal medium, Gcs1p was distributed over the cytoplasm in a fine punctate pattern with more concentrated staining in the perinuclear regions. Overexpressed Gcs1p in yeast was localized partially with mitochondria and partially in perinuclear structures close to mitochondria. The Gcs1p PH-domain was required for localization in mitochondria but not for the perinuclear region. Transport of carboxypeptidase Y and invertase was not significantly altered by disruption of the gcs1 gene. This mutation did, however, reduce mitochondrial lateral distribution and branching when yeast were grown in rich medium. In yeast overexpressing Gcs1p, mitochondrial morphology was aberrant, with unbranched tubules and large spherical structures. We suggest that Gcs1p may be involved in the maintenance of mitochondrial morphology, possibly through organizing the actin cytoskeleton in Saccharomyces.