Fluorescence flow cytometry was used to measure the internalization of the fluorescent ligand N-formyl-nle-leu-phe-nle-tyr-lys-fluorescein by human neutrophils. The internalization process was monitored by the accessibility of the receptor-bound fluorescent ligand to quenching following a change in the pH of the extracellular medium from 7.4 to 3.0. In such a pH change, extracellular ligand or fluorescein are quenched immediately (excitation 488 nm). In contrast, intracellular fluorescein (derived from fluorescein diacetate) or intracellular ligand are quenched with half-times of approximately 20 or approximately 40 sec, respectively, at 37 degrees C. The fraction of internalized ligand is calculated by resolving the fast and slow components of the quenching process. Temporal resolution of the internalization process in this system depends upon two factors. We have previously shown that it is possible to examine essentially continuously the kinetics of ligand binding in the nM concentration range without removing the free ligand (Sklar LA, Finney DA, Cytometry 3:161, 1982). We have now modified a Becton Dickinson FACS IV sample head assembly to permit direct addition of reagents into the cell suspension while on-line. This enables us to change the suspension pH and evaluate internalization with a time resolution of a few seconds. We observe that internalized ligand can be detected within 1 min and that the rate is proportional to the number of receptors occupied. The rate is essentially linear over the first few minutes and approximately 60% of the receptor-bound ligand is internalized after 3 min.