6-Phosphofructo-1-kinase (PFK-1) from a variety of species and organs can undergo phosphorylation by cAMP-dependent protein kinase. In most studies the stoichiometry of the phosphorylation reaction was far below the expected minimum value of 4 mol phosphate/mol PFK-1 tetramer. The present study with rat liver PFK-1 and purified catalytic subunit of cAMP-dependent protein kinase was undertaken in order to find the maximum phosphorylation stoichiometry under well-defined conditions. Irrespective of whether PFK-1 had been first treated with purified protein phosphatase 2C or not, no more than 1.66 +/- 0.22 mol phosphate/mol PFK-1 tetramer was incorporated, the highest single value being 2 mol phosphate/PFK-1 tetramer. This stoichiometry was found to be independent from the method of protein evaluation (gel dye-binding assay or amino acid analysis) and from the concentration of PFK-1 in the phosphorylation system (15.6 nM-0.53 microM). The stoichiometry was not affected by the presence of allosteric ligands, fructose-1,6-bisphosphatase or the PFK-1-inactivating protein. The possibility could be excluded that partial proteolysis was responsible for the incomplete phosphorylation. Two-dimensional polyacrylamide gel electrophoresis gave no indication of the existence of two different subunits in rat liver PFK-1. Possible reasons why rat liver PFK-1 undergoes 'half-of-the-sites' phosphorylation are discussed.