The rounding up of mitotic human Chang liver cells in monolayer culture was studied quantitatively. It was surprising to find significant reduction in cell surface area considering that endocytosis has been demonstrated to be at a complete standstill in M phase. Uptake studies using impermeant BCECF (2′,7′-bis(2-carboxyethyl)-4(5)-carboxyfluorescein free acid) pH indicator and particulate neutral red dye in aqueous buffer showed preferential internalization into mitotic cells in direct contrast to expectation since interphase cells do not have arrested endocytosis. However, infolded plasma membrane ruffles and internalized extracellular material were demonstrated in prophase cells, much like those seen in interphase rounding via the induction of intracellular alkalinizations. Raised intracellular pH (pHi) is a universal and consistent finding in M phase cells. Despite cessation of small pit endocytosis, it remains possible for plasma membrane internalization to be a causal factor in the observed surface area reduction in mitotic rounding. © 1993 Wiley-Liss, Inc.