When a cell is stimulated to perform a function in the body, an increase in the intracellular free concentrations of one or more molecules occurs first. These “”signaling”” molecules then switch on the various molecular machines that produce the desired function; such as muscle contraction, or formation of a memory. The processes by which signaling molecules are able to turn on only the correct machines are not understood. Disturbances in signaling are associated with many diseases; such as cancer, Altzheimer’s, arthritis, chronic pain and vascular hypertension. Ca2+ ion is a universal intracellular signaling molecule, and the Ca2+-binding protein calmodulin is responsible for converting Ca2+ signals into changes in the activities of a great many different molecular machines. Our work has focused on understanding both the cell biology and biochemistry of this key protein. We have recently begun investigations of Ca2+ and calmodulin function in the cell using genetically-encoded fluorescent indicator proteins developed in our laboratory. These allow us to follow the amounts of calmodulin-target complexes that are produced in different regions of an intact cell, so that we now can begin to develop an accurate picture of the factors determining how calmodulin, and the many molecular machines under its control, behave in the living organism.