Pool sizes of inositol phosphate species in myo-[3H]inositol-labeled porcine tracheal smooth muscle were determined under three conditions: (a) unstimulated; (b) stimulated with carbachol; (c) atropine-relaxed from a carbachol contraction. In unstimulated muscle, the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content was 14 pmol/100 nmol lipid P1. This is equivalent to a mean [Ins(1,4,5)P3] of about 3 microM (in total cellular water), a level about 30-fold in excess of that required for Ca2+ release from Ins(1,4,5)P3-sensitive sarcoplasmic reticulum (SR). Pool sizes of breakdown products of Ins(1,4,5)P3 were relatively small or absent in unstimulated muscle, suggesting that, under this condition, Ins(1,4,5)P3 was sequestered and had limited access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. During carbachol stimulation, the Ins(1,4,5)P3 pool did not increase while those of other mono-, di-, and trisphosphate isomers increased over 10-fold. Subsequent atropine-induced relaxation resulted in a partial depletion (40%) of total tissue Ins(1,4,5)P3. Decreases in Ins(1,4,5)P3 were paralleled by decreases in Ins(1,4)P2 and Ins(1,3,4)P3. During contraction a portion of total tissue Ins(1,4,5)P3 has access to Ins(1,4,5)P3 3-kinase and 5-phosphatase and to Ins(1,4,5)P3-sensitive SR, though during antagonist-induced relaxation access to Ins(1,4,5)P3-sensitive SR for Ca2+ release is restricted. Data are consistent with a mechanism by which a large pool of Ins(1,4,5)P3 present in the unstimulated state in a sequestered compartment can contribute in activated muscle to increases in [Ins(1,4,5)P3] in a nonsequestered compartment, controlling SR Ca2+ release.