Autofluorescence of cells can be a major portion of the fluorescence signal in many systems, especially when fluorescent conjugates are used to study receptor-ligand systems for which there are less than 70,000 receptors per cell. We have devised a method for the cell-by-cell correction of autofluorescence for flow cytometric data by using an additional parameter to measure and correct for autofluorescence in the fluorescence channel. The principle has been extended to allow simultaneous correction for autofluorescence and dual-fluorescence spillover compensation in samples labeled with two different fluorochromes; all corrections were done in software, making them applicable to any flow cytometer. The autofluorescence correction method was used to analyze the acidification of epidermal growth factor (EGF) by Swiss 3T3 cells. EGF is acidified to pH 6.2 starting two min after labeling, with a half-time for acidification of 45 s.