The phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (EC 2.7.1.105/ EC 3.1.3.46) in rosette leaves of Arabidopsis was examined. Immunoblotting with specific antisera detected 96-kDa and 92-kDa bands in the crude protein extracts from rosette leaves of Arabidopsis. Incubation of protein samples with alkaline phosphatase before SDS-PAGE reduced the 96-kDa band with concomitant increase of the 92-kDa band, suggesting that the former is a phosphorylated form of the latter. In accordance with this result, 96-kDa and 92-kDa bands were immuno-precipitated from the crude protein extracts from [(32)P]orthophosphate-labeled rosettes of Arabidopsis; and, the former was heavily labeled, the latter faintly labeled. Analysis of phospho-amino acid residues derived from the [(32)P]-labeled 96-kDa band revealed that the phosphorylation occurred on serine and threonine residues, excluding the possibility that the phosphorylated band represent a phospho-histidine intermediate that is known to form in the phosphatase reaction. The relative level of the 96-kDa band over the 92-kDa band in whole rosette extracts changed diurnally and was highest at the beginning of nighttime. Furthermore, the 96-kDa band was highly enriched in the extracts of very young rosette leaves, suggesting that the phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase is regulated physiologically and developmentally in Arabidopsis.