HeLa S-3 cells held in metaphase with colcemid or vinblastine sulphate (VLB) for up to 6 h developed confronting cisternae (CCs) in their protoplasm. They were first detected about 1 h after exposure to alkaloid began and became progressively more prominent until by 6 h they could be found in almost every metaphase cell. As time elapsed, there was also an increasing incidence of multiple stacking of CCs. The progressive development of CCs with length of alkaloid exposure was mirrored by a corresponding reduction of rough endoplasmic reticulum (rER) in the protoplasm of the arrested metaphases. In contrast, an equally extensive survey of HeLa S-3 metaphases arrested with nocodazole revealed only one poor example of a CC-like structure. CCs were not found in a) normal mitotic cells, b) interphase cells whether or not they had been exposed to alkaloids, c) cells arrested in metaphase for some period of time by incubation of cells at 27 degrees C for 3 days, and d) large polyploid metaphases which had spent long times progressing through division. In a brief survey of three other cell lines before and after colcemid treatment (3T3, L-132, and B16F10 cells), CCs were encountered relatively infrequently in the colcemid exposed cells, even after 6 h exposure. The relationship of CCs to other cell membrane systems, and their appearance relative to those previously reported in neoplastic and virus-infected cells are discussed.