The correlation between acetylcholine induced changes in the intracellular free, Ca2+ concentration ([Ca2+]i), and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in isolated acini from the rat parotid and lacrimal glands was investigated. Applying digital image processing on Fura-2 loaded acini, we observed that Ca2+ increases either simultaneously throughout the acinar configurations or that occasionally, the rise near the lumen can precede the rise near the basal part by 50-100 ms. Measurements on cell suspensions revealed a correlation between changes in [Ca2+]i and changes in the cellular Ins(1,4,5)P3 content, and it is concluded that in the individual cells Ins(1,4,5)P3 is released to the cytosol within the first second after stimulation. Applying a diffusion coefficient for cytoplasmic Ins(1,4,5)P3 of 2.83 x 10(-6) cm2/s (Allbritton et al., 1992, Science, 258, 1812-1815), we have calculated the concentration profile for this messenger in a sphere with a radius of 10 microns where Ins(1,4,5)P3 is released in the center following a monoexponential function with a rate constant of 4 s-1. Assuming that Ins(1,4,5)P3 concentrations of 1 or 5% of the maximum value is able to release Ca2+, we calculated that Ca2+ waves can appear at a rate of 100 or 40 microns/s. The present data are consistent with Ins(1,4,5)P3 being a cellular messenger, that by diffusion, initiates the Ca2+ release from the cellular pools within the first fraction of a second.