Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPβ localization in mammalian cells. We demonstrate PITPβ localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPβ. Domain mapping analyses show the targeting information within PITPβ consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W202W203 motif). Combination of the specificity elements with the W202W203 motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPβ association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPβ isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPβ localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms δ or ε. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser 262 is required for PITPβ targeting to Golgi membranes. © 2006 by The American Society for Cell Biology.