Inosine diphosphatase of a Golgi-enriched fraction of the onion root tip was characterized. Peak enzyme activity occurred at pH 4.8 and 7.0, although considerable activity was present between the peaks. The activity at neutral pH approximately doubled during a 4-day cold storage; no such increase of the pH 4.8 activity occurred under similar conditions. Treatment with deoxycholate or Triton X-l00 also activated the pH 7.0 enzyme. Although magnesium, manganese and calcium all supported inosine diphosphatase activity, 2 mM manganese supported maximal activity. Uridine diphosphate, guanosine diphosphate and inosine diphosphate were hydrolyzed much more rapidly than the other substrates tested. Sodium fluoride, uranyl nitrate, potassium chloride and heat all partially inhibited the enzyme. Glutaraldehyde and lead nitrate, two reagents to which nucleoside diphosphatase is exposed when studied cytochemically, greatly reduced enzyme activity.