The effect of temperature on the kinetics of endocytosis by B lymphocytes and BCL1 cells was examined by using flow cytometry. Mouse B cells were stained with FITC-R alpha MIg and induced to undergo cap formation in the temperature-controlled sample compartment of the flow cytometer. Capping and subsequent endocytosis were measured by continually monitoring the pulse profile descriptors (width and area) of the electronic signal curves generated during flow cytometric analyses. Decreases in area values which immediately followed cap formation were shown to result from a pH-dependent quenching of fluorescence emission as the internalized FITC-R alpha MIg entered acidic subcellular compartments. Similar results were obtained with BCL1 cells, but, in addition to cap formation and acidification, cytoplasmic diffusion of the fluorescent ligand could also be discerned by flow cytometry. With either cell type the rates of cap formation and endocytosis were shown to be temperature dependent with temperature coefficient (Q10) values of 2.0-2.7. Based upon Arrhenius plots of width and area changes, activation energies for capping and endocytosis ranged from approximately 12-18 kcal/mol.