Studies were undertaken to provide a systematic investigation of the effect of the chemotactic factor, formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe), on the levels of total and exchangeable calcium in rabbit neutrophils. The 45Ca2+ specific activity of the cells was thus determined before and after stimulation by chemotactic factors. Total cell calcium was found to be constant in neutrophils equilibrated with 50-1,000 microM extracellular calcium but decreased by 30% at an extracellular calcium concentration of 5 microM. F-Met-Leu-Phe had little, if any, statistically significant effect on the level of total cell calcium. In contrast, F-Met-Leu-Phe affects greatly the steady-state levels of radioactive calcium in the neutrophils. The primary effect is an initial loss followed by an increase of cell-associated radiolabeled calcium. The extent and even the direction of the effect depends on the level of extracellular calcium as well as the concentration of the chemotactic factor and the duration of its interaction with the cell. The results are discussed in terms of the hypothesis that the binding of the chemotactic factor to its receptor leads to, among other things, a graded displacement of previously bound Ca2+.