We have previously reported that rearward migration of surface particles on slowly moving cells is not driven by membrane flow (Sheetz, M. P., S. Turney, H. Qian, and E. L. Elson. 1989. Nature (Lond.). 340:284-288) and recent photobleaching measurements have ruled out any rapid rearward lipid flow (Lee, J., M. Gustafsson, D. E. Magnussen, and K. Jacobson. 1990. Science (Wash. DC.) 247:1229-1233). It was not possible, however, to conclude from those studies that a slower or tank-tread membrane lipid flow does not occur. Therefore, we have used the technology of single particle tracking to examine the movements of diffusing particles on rapidly locomoting fish keratocytes where the membrane current is likely to be greatest. The keratocytes had a smooth lamellipodial surface on which bound Con A-coated gold particles were observed either to track toward the nuclear region (velocity of 0.35 ± 0.15 μm/s) or to diffuse randomly (apparent diffusion coefficient of [3.5 ± 2.0] × 10-2 cm2/s). We detected no systematic drift relative to the cell edge of particles undergoing random diffusion even after the cell had moved many micrometers. The average net particle displacement was 0.01 ± 2.7% of the cell displacement. These results strongly suggest that neither the motions of membrane proteins driven by the cytoskeleton nor other possible factors produce a bulk flow of membrane lipid.