An immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5-bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd-incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 X 10(5) cells can be fixed, hydrolyzed and treated with the anti-BrdUrd antiserum. Nuclear-bound IgG is localized by fluorescein-labeled avidin-D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of "labeled" cells can be followed through the cell cycle.