Unimbibed, mature cotton seeds (cv. Deltapine 61) were found to possess activity for all gluconeogenesis-related enzymes examined, except for isocitrate lyase activity. This indicates that transcription and translation of most enzymes needed for postgerminative growth takes place during seed maturation. This is in contrast with the generalization that "germination" enzymes are synthesized de novo from previously untranslated mRNAs conserved in dry seeds. AB enzyme activities increased 3-fold or greater following imbibition, and most remained constant after reaching their peak. Notable exceptions were activities for three /t oxidation enzymes and fructose bisphosphatase, which decreased precipitously after peaking with other enzyme activities.Standard (2, 17-19, 21, 26), it became important to determine which gluconeogenesis-related enzymes were already present in unimbibed seeds, and where they were compartmentalized within cells during postgerminative growth. We attempted to isolate glyoxysomes on sucrose gradients in swing-out rotors according to established procedures, but found these methods were unsuitable for separating cotton glyoxysomes from other organelles.In this paper, we show the activity of numerous gluconeogenesis-related enzymes in unimbibed seeds, and tabulate development of these activities in cotyledons as lipid reserves are mobilized. A reliable procedure is described for isolating and purifying cotton glyoxysomes via isopycnic centrifugation in a zonal rotor.