Previous studies indicated that activation of alpha 1-adrenergic receptors in BC3H-1 muscle cells (S. K. Ambler and P. Taylor, J. Biol. Chem. 261:5866-5871, 1986) and muscarinic receptors in 1321N1 astrocytoma cells (S. B. Masters, T. K. Harden, and J. H. Brown, Mol. Pharmacol. 27:325-332, 1985) resulted in the rapid mobilization of Ca2+ from internal stores of both cell types. Paradoxically, alpha 1-adrenergic agonists did not rapidly increase inositol trisphosphate (Ins-P3) formation in BC3H-1 cells, in distinction to the rapid increase in Ins-P3 accumulation observed in 1321N1 cells after muscarinic stimulation. To determine whether the variations observed in the Ins-P3 response could be ascribed to differences in the relative amounts of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol tetrakisphosphate (respectively, Ins-1,4,5-P3, Ins-1,3,4-P3, and Ins-P4), we have separated the individual inositol phosphates by high-performance liquid chromatography and examined the rates of conversion of individual inositol phosphates in the two types of cells. Muscarinic stimulation of 1321N1 cells resulted in increased Ins-1,4,5-P3 production, as well as the rapid production of Ins-1,3,4-P3 and Ins-P4. Application of alpha 1-agonist to BC3H-1 cells produced a modest but delayed increase in accumulation of Ins-1,4,5-P3. Adrenergic stimulation also resulted in a smaller and even slower production of Ins-1,3,4-P3, and Ins-P4 could not be detected in BC3H-1 cells under any conditions employed. Thus, over a 30-sec interval in which Ca2+ is mobilized to a maximum extent, increases in Ins-1,4,5-P3, Ins-1,3,4-P3, or Ins-P4 amounted to less than 10% over basal values in BC3H-1 cells. These results indicate that the regulation of Ins-P3 isomer formation and conversion may vary substantially between different cell types. In addition, if inositol 1,4,5-trisphosphate is the sole mediator of intracellular Ca2+ release, it is necessary to propose that an increase in Ins-1,4,5-P3 sufficient to mobilize Ca2+ rapidly may occur only within discrete cellular localities in some cell types. According, it may not be possible to detect the increases in Ins-1,4,5-P3 over basal concentrations when measuring total cellular inositol phosphates.