SnRK1 of the SNF1/AMPK group of protein kinases is an important regulatory protein kinase in plants. SnRK1 was recently shown as a target of the sugar signal, trehalose 6-phosphate (T6P). Glucose 6-phosphate (G6P) can also inhibit SnRK1 and given the similarity in structure to T6P, we sought to establish if each could impart distinct inhibition of SnRK1. Other central metabolites, glucose 1-phosphate (G1P), fructose 6-phosphate and UDP-glucose were also tested, and additionally ribose 5-phosphate (R5P), recently reported to inhibit SnRK1 strongly in wheat grain tissue. For the metabolites that inhibited SnRK1, kinetic models show that T6P, G1P and G6P each provide distinct regulation (50% inhibition of SnRK1 at 5.4 mu M, 480 mu M, >1 mM, respectively). Strikingly, G1P in combination with T6P inhibited SnRK1 synergistically. R5P, in contrast to the other inhibitors, inhibited SnRK1 in green tissues only. We show that this is due to consumption of ATP in the assay mediated by phosphoribulokinase during conversion of R5P to ribulose-1,5-bisphosphate. The accompanying loss of ATP limits the activity of SnRK1 giving rise to an apparent inhibition of SnRK1. Inhibition of SnRK1 by R5P in wheat grain preparations can be explained by the presence of green pericarp tissue; this exposes an important caveat in the assessment of potential protein kinase inhibitors. Data provide further insight into the regulation of SnRK1 by metabolites. Crown Copyright (c) 2012 Published by Elsevier Masson SAS. All rights reserved.