Chlorophyll fluorescence at short and long wavelengths was used to sort thylakoid membranes of maize, a plant with the C4 dicarboxylic acid pathway of photosynthesis, in a flow cytometer. The method yielded two distinct particle populations that were identified as mesophyll and bundle sheath thylakoids by low-temperature fluorescence spectroscopy and by the pigment ratio of chlorophyll a/b. Mesophyll and bundle sheath thylakoids were essentially pure after sorting by flow cytometry. Fluorescence data and chlorophyll a/b pigment ratios of thylakoids separated by flow cytometry were compared with earlier data of preparations obtained by conventional isolation procedures. Our results indicate that impure mesophyll and bundle sheath membranes were used in most previous investigations. We were unable to detect the major light-harvesting complex of PS II (LHC II) in our pure bundle sheath thylakoids using fluorescence excitation spectroscopy. Therefore, we believe that the previously reported presence of LHC II in bundle sheath chloroplasts of maize can be attributed to mesophyll contamination.