We have examined the time course of the formation of inositol mono-, bis-, tris, and tetrakisphosphates (InsP1, InsP2, InsP3, and InsP4, respectively) in slices of the longitudinal muscle of guinea pig small intestine that had been prelabeled with myo-3H-inositol. The agonists employed were histamine and carbachol. InsP3 increases immediately with a time course which is similar to that of the increase in contractile force and remains elevated for the rest of the incubation period. High performance liquid chromatography analysis revealed that InsP3 is composed of two isomers, the 1,4,5- and 1,3,4-isomers. The release of 1,4,5-inositol trisphosphate [Ins(1,4,5)P3] was followed by the rapid accumulation of InsP4 and later on by the formation of 1,3,4-inositol trisphosphate [Ins(1,3,4)P3]. Ins(1,3,4)P3 and InsP4 were identified by co-chromatography with the Ins(1,3,4)P3 and 1,3,4,5-inositol tetrakisphosphate prepared from 3H-Ins(1,4,5)P3 using a kinase from rat brain. The time course of accumulation of these compounds is consistent with a second messenger role of Ins(1,4,5)P3 in initiation of smooth muscle contraction.