Exocytosis in yeast occurs at plasma membrane subdomains whose locations vary with the cell cycle, but the primary protein determinants of these sites are unknown. A functional fusion of Sec3 protein with green fluorescent protein (Sec3-GFP) localizes to the site of polarized exocytosis for each cell-cycle stage, where it colocalizes with Sec4p and Sec8p. Sec3-GFP localization is independent of secretory pathway function, of the actin and septin cytoskeletons, and of the polarity establishment proteins. We propose that Sec3p is a spatial landmark defining sites of exocytosis. Polarized secretion would result from the coupling of actin-dependent vesicle targeting with Sec3p-dependent establishment of the vesicle fusion site.