The hormonal regulation and molecular forms of Type L pyruvate kinase were investigated in rat hepatocytes maintained in primary culture. Five isoelectric forms of the enzyme subunit were identified by isoelectric focusing in 8 M urea. Immediately after pulse labeling rat hepatocytes with [35S]methionine radioactivity was observed in one major (D-band) and one minor (I-band) peptide band. These isoelectric forms were shown to be dephosphorylated forms of the subunit. Acute administration of 0.1 microM glucagon was accompanied by disappearance of the D- and I-bands and appearance of two additional forms (P- and A-bands, respectively). These latter two forms were demonstrated to be phosphorylated forms of the subunit. A fifth isoelectric form of the pyruvate kinase subunit (B-band) was identified by immunolocation; however, incorporation of radioisotope into this band was low. Chronic administration of glucagon or dexamethasone had no significant influence on the molecular properties of pyruvate kinase. However, novel observations concerning the influence of glucose and ethanol on the phosphorylation state of the enzyme were made. When hepatocytes were maintained at 5.5 mM glucose for 24-48 h, the activity ratio for pyruvate kinase decreased from 0.65 to 0.40 and the enzyme became partially phosphorylated. Raising the glucose concentration to 28 mM prevented or rapidly reversed the phosphorylation state of the enzyme. Administration of low concentrations of ethanol (1-20 mM) caused a decline in the activity ratio of pyruvate kinase in the presence of both 5.5 and 28 mM glucose. These latter observations concerning the influence of glucose and ethanol are the first demonstrating that nutrients or metabolites alter the phosphorylation state of the enzyme in the absence of hormonal stimuli.