In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Alabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. All three kinases gave phosphopeptide CNBr-cleavage maps of HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arro, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+ dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PKI is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on-gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-independent, inactivated by PP2A or PP2C, had a require ment for a hydrophobic residue in the. +4 position of peptide substrates, had a molecular mass by gel filtration of similar to 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognised a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported previously for the SNF1-like enzyme, HRK-A. Our results indicate a considerable complexity of kinase cascades mediating the regulation of assimilatory and biosynthetic pathways in response to environmental stimuli in plants.