The rich heterogeneity of renal tubular membranes and cells continues to provide formidable challenges in the isolation of homogeneous membrane vesicle populations for study. The present study applies flow cytometry, the technique of fluorescence-activated cell sorting, to the study of brush border membrane vesicles. Direct comparison was made of enzymatic marker purity of rat renal cortical brush border membrane vesicles prepared by divalent ion precipitation, or flow cytometry sorting. Flow cytometry sorted membrane vesicles were characterized by greater brush border membrane markers, no detectable mitochondrial or basolateral markers, and greatly reduced Golgi and lysosomal markers. The flow sorted membrane vesicles were functional for transport studies as they took up at least as much 3H-proline and 3H-glucose per mg protein as divalent ion precipitation membrane vesicles. Preparation of membrane vesicles from superficial and deep cortex allowed us to image the different distributions of gamma-glutamyl transferase in membrane vesicles from these areas. Hence, membrane vesicle populations of exceptional purity can be separated according to fluorescent markers using flow cytometry. High speed observations on large numbers of individual vesicles allows identification of subpopulations, and statistical comparison, within a single heterogeneous sample.