Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14p(K66,239A) is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14p(K66,239A) fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1(ts) and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.