A second fructokinase (EC 184.108.40.206) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K(m) values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K(m) for Mg adenosine 5'-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg(2+) was essential for activity but the enzyme was inhibited by excess Mg(2+) or ATP. Mg adenosine 5'-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K(+), Rb(+), and NH(4) (+) were the most effective. The possible role of fructokinase (fraction III) is discussed.