Cultured human erythroleukemia (HEL) cells were used to study the genesis of polarity in single cells. HEL cells grow in suspension in culture medium, but attach and spread on fibronectin when treated with 10 nM phorbol myristate acetate. If the spread cells are treated with dibutyryl cyclic adenosine monophosphate, about 50% of the cells polarize and form very striking elongated processes. Time‐lapse video microscopy showed that elongation develops in these cells because the anterior pole of the cell, which bears a small ruffled membrane, moves slowly (approximately 0.16 μm/min) forward on the substratum elongating the posterior pole or tail behind it. Using indirect immunofluorescence we found that elongation of the tail correlates with the development of long microtubule bundles emanating from the centrosome, which is located posterior to the nucleus on the trailing side of the cell. Incubation with nocodazole, which inhibited development of the long microtubules and the elongation, resulted in a centrosome positioned over the nucleus in 45% of the cells and extension of the membrane ruffling to many points around the cell's periphery. Unexpectedly, time‐lapse video microscopy demonstrated that the treated cultures also contained some smaller cells with very marked anterior ruffles and short tails. These cells moved rapidly about the culture dish (maximum 0.8 μm/min: average 0.5 μm/min). In these fast moving cells the centrosome was also located posterior to the nucleus. Several recent reports have stressed the importance of relocation of the centrosome to an anterior position in cells developing polarity after experimental wounding. Our results show that both striking polarization and rapid motility can occur without such a relocation. The polarity induced in the HEL cells correlates most clearly with the limitation of membrane ruffling to one region; this limitation is removed by microtubule disassembly. We therefore propose that localized ruffling is the critical first step in polarized motility generally, and that centrosomal position is related to other factors. Cell Motil. Cytoskeleton 36:203–215, 1997. © 1997 Wiley‐Liss, Inc.