Glycogen storage disease type Ib is caused by deficiencies in the glucose-6-phosphate transporter (G6PT), a phosphate (P(i))-linked antiporter capable of homologous (P(i):P(i)) and heterologous (G6P:P(i)) exchanges similar to the bacterial hexose-6-phosphate transporter, UhpT. Protease protection and glycosylation scanning assays have suggested that G6PT is anchored to the endoplasmic reticulum by 10 transmembrane domains. However, recent homology modeling proposed that G6PT may contain 12 helices and that amino acids essential for the functions of UhpT also play important roles in G6PT. Site-directed mutagenesis and in vitro expression assays demonstrated that only one of the four residues critical for UhpT activity is essential in G6PT. Furthermore, glycosylation scanning and protease sensitivity assays showed that the 10-domain model of G6PT is more probable than the 12-domain UhpT-like model.