A kinetic analysis of exchangeable calcium associated with rat anterior pituitary slices was carried out. Slices were permitted to accumulate 45Ca for 1 h, and then a desaturation experiment was initiated. The resulting data was represented as a polyexponential function and analyzed with a weighted nonlinear least-squares method. The resulting coefficients and exponents were used to determine, at the steady state, the size of each calcium compartment, the unidirectional fluxes into and out of each compartment, and the corresponding rat constants. Three kinetically distinguishable compartments were defined. The first compartment exchanges with a half-time slower than calcium free in the extracellular space and can be removed by a 1.5-min exposure to Ca-free EGTA media suggesting that this calcium is bound in the extracellular matrix perhaps to the cell surfaces. The second compartment is intracellular and does not contain calcium accumulating structures such as mitochondria or microsomes. The third compartment has many of the characteristics associated with these organelles. Experiments were performed with two pituitary secretagogues, elevated K+ and ouabain, to assess changes in the study-state calcium distribution associated with enhanced secretion.