Two different hexose-phosphorylating enzymes, hexokinase and fructokinase, were partially purified from suspension-cultured Catharanthus roseus cells. One of the enzymes, hexokinase, catalyzed the phosphorylation of both glucose and fructose. The K m values for glucose and fructose were 0.06 mM and 0.23 mM, respectively. The V max of the enzyme with fructose was approximately three times higher than with glucose. This enzyme was specific in its requirement for ATP and its K m value for ATP was 52 μM. The optimum pH was 8.0 and Mg 2+ or Mn 2+ was required for the activity. The activity was inhibited by considerably higher concentrations of ADP (i.e., 4 mM ADP was required for 50% inhibition). The second enzyme, fructokinase, was specific for fructose, and no activity was detected with glucose as substrate. This enzyme used UTP or CTP as phosphate donor. The K m values of this enzyme for fructose and UTP were 0.13 mM and 0.15 mM, respectively. The pH optimum was 7.2, and Mg 2+ or Mn 2+ was required for the activity. These divalent cations could be partially replaced by Ca 2+ . The activity was inhibited noncompetitively by ADP and AMP. 90% inhibition of the activity by 0.5 mM ADP was observed in the presence of 2 mM UTP and 5 mM MgCl 2 . Fructose-2,6-bisphosphate, glucose-1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate had little or no effect on the activity of both the hexokinase and the fructokinase. Based on these results, a discussion is presented of the role of hexokinase and fructokinase and their involvement in the regulation of the metabolism of sugars in Catharanthus cells.