Phosphofructokinase (PFK) from Dictyostelium discoideum is a non-allosteric enzyme that lacks any of the characteristic regulatory mechanisms of PFK from other cells. We have determined the DNA sequence and analyzed the amino acid sequence of D. discoideum PFK, as an initial step toward understanding the peculiar properties of this enzyme. Three overlapping fragments, two of cDNA and one of genomic DNA, were isolated, which together could encode the complete sequence of D. discoideum PFK. The constructed full-length cDNA coded for a protein of 834 amino acids, with a calculated molecular mass of 92.4 kDa, which was similar to other eukaryotic and prokaryotic PFK. Alignments of the amino acid sequence with other isozymes revealed that many of the amino acid residues assigned to binding sites of substrates and allosteric effectors are conserved in this enzyme, but changes were also found that may contribute to the absence of allosteric mechanisms. A phylogenetic tree for the eukaryotic PFK family was constructed and showed that the N-terminal domain clustered with those of yeast subunits, whereas the C-terminal domain was more related to PFK from metazoa. Southern blotting indicated that D. discoideum PFK is encoded by a single gene. The enzyme is present throughout the life cycle of D. discoideum, with a gradual decrease of its expression during development.