Sialic acids are sometimes 9-O-acetylated in a developmentally regulated and cell-type-specific manner. Cells naturally expressing the disialoganglioside GD3 often O-acetylate the terminal sialic acid residue, giving 9-O-acetyl-GD3 (9AcGD3), a marker of neural differentiation and malignant transformation. We also reported that Chinese hamster ovary cells transfected with GD3 synthase can spontaneously O-acetylate some of the newly synthesized GD3. It is unclear whether such phenomena result from induction of the 9-O-acetylation machinery and whether induction is caused by the GD3 synthase protein or by the GD3 molecule itself. We now show that exogenously added GD3 rapidly incorporates into the plasma membrane of Chinese hamster ovary cells, and 9AcGD3 is detected after ∼6 h. The incorporated GD3 and newly synthesized 9AcGD3 have a half-life of ∼24 h. This phenomenon is also seen in other cell types, such as human diploid fibroblasts. Inhibitors of gene transcription, protein translation, or endoplasmic reticulum-to-Golgi transport each prevent induction of 9-O-acetylation, without affecting GD3 incorporation. Inhibition of the initial clathrin-independent internalization of incorporated GD3 also blocks induction of 9-O-acetylation. Thus, new synthesis of one or more components of the 9-O-acetylation machinery is induced by incorporation and internalization of GD3. Prepriming with structurally related gangliosides fails to accelerate the onset of 9-O-acetylation of subsequently added GD3, indicating a requirement for specific recognition of GD3. To our knowledge, this is the first example wherein a newly expressed or exogenously introduced ganglioside induces de novo synthesis of an enzymatic machinery to modify itself, and the first evidence for a mechanism of induction of sialic acid O-acetylation. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.