Stimulation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity and accelerated growth occur in cultures of monkey kidney epithelial cells (BSC-1 line) that are exposed to medium with a reduced K concentration (3.2 mM). We recently found that this activation of G3PD was mediated by the appearance of a new cytosolic protein with an apparent molecular weight of 62,000. G3PD and this modifier protein were isolated from BSC-1 cells, and the interaction between them was characterized to define the mechanism(s) of enzyme activation. The enzyme protein was purified from cells grown in control medium (5.4 mM K). The enzyme, in the presence of modifier, exhibited an increase in maximal rate of enzyme reaction and a decrease in the apparent Km for NAD+. Analysis using Dixon plots revealed that the presence of modifier increased the Ki for NADH by two- to threefold. Inhibition by NADH was competitive with respect to NAD+, glyceraldehyde-3-phosphate, and inorganic phosphate. ATP also inhibited enzyme activity in a competitive manner with respect to NAD+; however, the Ki for ATP was similar both in the presence and absence of modifier. These results suggest that one mechanism by which the cytosolic modifier protein stimulates G3PD activity is to decrease product inhibition by NADH.