AbstractKinetic analysis of PFK‐1 from rodent AS‐30D, and human HeLa and MCF‐7 carcinomas revealed sigmoidal [fructose 6‐phosphate, Fru6P]‐rate curves with different Vm values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH, or K+. The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non‐essential hyperbolic activation. PFK‐1 from rat liver and heart also exhibited the mixed cooperative‐hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased Vm and increased content of inactive (T) enzyme forms. High K+ prompted increased (Fru6P) or decreased (activators) affinities; increased Vm; and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK‐1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF‐7 cells, of L > M in AS‐30D cells, and of C in HeLa cells. PFK‐1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK‐1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK‐1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK‐1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux‐control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect. J. Cell. Biochem. 113: 1692–1703, 2012. © 2011 Wiley Periodicals, Inc.