Further properties and the mechanism of action of homogeneous inositol l-phosphate synthase (EC 5.5.1.4) from rat testis have been examined. The enzyme is maximally active at pH 7.7. Relative to basal activity measured in the presence of Tris ion (50 mM) and NAD ion (1 mM) as sole cations, the addition of 1 mM K ion or NH4 ion stimulates the activity 275 and 400%, respectively. Both cations decrease Km and increase V-max markedly for both substrate (glucose 6-phosphate) and cofactor (NAD ion). Li ion also decreases Km but anomalously decreases V-max for glucose 6-phosphate. Heavy metals inhibit completely, Zn2 ion and Cd2 ion reversibly and Cu2 ion irreversibly. EDTA up to 100 mM has no effect on basal activity suggesting no metal requirement and eliminating the enzyme as a Class II aldolase. Failure of NaBH4 reduction to inactivate the enzyme and to bind label in the presence of labeled substrate rules out a Schiff base mechanism eliminating the enzyme as a Class I aldolase.