Intact granules were isolated from sonicated purified rat serosal mast cells on a Percoll gradient. The granules were demonstrated to contain a diphosphoinositide kinase that catalyzes the formation of triphosphoinositide from diphosphoinositide. The enzyme requires adenosine triphosphate and Mg2+ or Mn2+ for activity. The Km for adenosine triphosphate is 3 mumol/L, and maximal response is observed at 20 mmol/L of Mg2+ or 1 mmol/L of Mn2+, respectively. Triphosphoinositide synthesis in the granules is dependent on the time and temperature of the incubations. Cyclic adenosine monophosphate, adenosine, adenosine diphosphate, and adenosine monophosphate decrease the enzyme activity. A comparison of the rate of phosphorylation of intact and broken membrane granules suggests that the phosphorylation occurs on the outer (cytoplasmic) surface of the granules.