Eukaryotic cells consume glucose through glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. Glycolytic intermediates can also be converted into ribose by enzymes in the nonoxidative arm of the pathway, the regulation of which is poorly understood. By carrying out a metabolomics screen of yeast deletion mutations of genes of unknown function, Clasquin et al. found that deletion of SHB17 causes the accumulation and depletion of certain metabolites. Biochemical assays helped determine the endogenous substrates of Shb17, sedoheptulose-1,7-bisphosphate (SBP) and octulose-1,8-bisphosphate (OBP), which are converted into sedoheptulose-7-phosphate (S7P) and octulose-8-phosphate (O8P), respectively. Shb17 had previously been shown to have phosphatase activity against the structurally similar metabolite fructose-1,6-bisphosphate (FBP) in vitro, but FBP does not accumulate in the SHB17 deletion strain, and kinetic studies confirmed Shb17's preference for SBP. Structural analysis of the Shb17–SBP complex revealed strong similarities to the recently determined structure of Shb17 in complex with FBP. However, Shb17 makes additional hydrogen bond interactions with SBP, and SBP binds the active site in a more favorable closed furan conformation, which may explain its higher affinity. Isotope labeling studies allowed the quantification of carbon-to-ribose flux: flux through Shb17 increases when ribose demand is high relative to the demand for NADPH. In metabolically synchronized yeast cells, Shb17 expression levels are correlated with expression levels of ribosomal proteins, suggesting that periodic Shb17 expression coincides with the peak demand for ribose phosphate that occurs during ribosome synthesis. Together, these findings suggest that Shb17 links the pentose phosphate pathway and glycolysis in a sequence of reactions—called riboneogenesis—by catalyzing a strongly thermodynamically driven dephosphorylation step. Through this process, the riboneogenesis pathway converts glycolytic intermediates into ribose-5-phosphate without the production of NADPH, allowing the cell to adjust the flux of carbon to ribose in response to changing conditions. (Cell 145, 969–980, 2011)