To understand the insulin-induced activation of 6-phosphofructo-2-kinase (PFK-2) of the bifunctional enzyme PFK-2/fructose-2,6-bisphosphatase in heart, the effect of phosphorylation by protein kinases of the insulin signaling pathways on PFK-2 activity was studied. Purified PFK-2/fructose-2,6-bisphosphatase from bovine heart is a mixture of two isoforms (M r 58,000 and 54,000 on SDS-polyacrylamide gels). The M r 54,000 protein is an alternatively spliced form, lacking phosphorylation sites for protein kinases. Recombinant enzymes corresponding to the M r 58,000 (BH1) and M r 54,000 (BH3) forms were expressed and used as substrates for phosphorylation. The recombinant BH1 isoform was phosphorylated by p70 ribosomal S6 kinase (p70 s6k ), mitogenactivated protein kinase-activated protein kinase-1, and protein kinase B (PKB), whereas the recombinant BH3 isoform was a poor substrate for these protein kinases. Treatment with all protein kinases activated PFK-2 in the recombinant BH1 preparation. Phosphorylation of the recombinant BH1 isoform correlated with PFK-2 activation and was reversed by treatment with protein phosphatase 2A. All the protein kinases phosphorylated Ser-466 and Ser-483 in the BH1 isoform, but to different extents: p70 s6k preferentially phosphorylated Ser-466, whereas mitogen-activated protein kinase-activated protein kinase-1 and PKB phosphorylated Ser-466 and Ser-483 to a similar extent. We propose that PKB is part of the insulin signaling cascade for PFK-2 activation in heart.