ABSTRACT Synchronous populations of mitotic HeLa cells were obtained by the controlled agitation method, and a detailed morphological study of the cells in all phases of the cell cycle was under-taken to correlate variations in cell structures to known coexisting biochemical events. Auto-radiographic techniques using tritiated thymidine (3H-TdR) were used to detect S1 cells, and colcemid was added to some G2 samples to prevent the cells from going into the next cycle, thus preventing contamination with G1 cells. The approximate duration (in hours) of the 4 phases were as follows: M = 1·4, G1= 8–9, S = 7, G2 = 4, and the generation time (T) = 21 ± 2 h. Randomization of the cell populations became apparent in the G2 phase (contaminated with S and M cells) and was most likely a result of the genetic make-up of the individual (mixoploid) HeLa cells, nutritional factors (serum lots used), temperature shock, and handling effects. Polyribosomes shifted to monomeric ribosomes during late prophase, at which time nucleoli also break down. These changes are correlated with the drop in protein and RNA synthesis reported for mitotic mammalian cells. The Golgi apparatus persisted in a modified form throughout mitosis. The mid-body forms from the anaphase stem-body and may interfere with the separation of daughter cells. Our studies suggest that the mid-body goes to one of the daughter cells where remnants of this structure were seen in early G1 cells. Large numbers of polyribosomes and the presence of well-developed nucleoli (many attached to the nuclear envelope) characterized G1. These structures, which play a major role in protein and RNA synthesis, persist with slight variations throughout inter-phase. Dense fibrillar nuclear bodies were prominent in late G1 cells. Centrioles separate during G1, and replicate by orthogonal budding during the S phase. Reproducible labelling patterns which reflect the asynchronous multireplicon nature of DNA synthesis in mammalian cells were characteristic of the various stages of the 51 phase. Granular nuclear bodies, which were prominent in S and G2 cells, may correspond to the larger species of heterogeneous nuclear RNA found in HeLa cells. G2 cells were similar in appearance to S cells. As heterochromatin areas increased in late G2 and prophase, perichromatin granules (of unknown significance) became prominent. Mitochondria behaved as independent cell organelles throughout the cell cycle. Hypertrophied RER, SER, and annulate lamellae, characterized the cytoplasm of colcemid-treated cells. The above changes are indicative of increased metabolic activity, and these structures may function in the production of colcemid-detoxifying enzymes in a manner analogous to that of drug-treated hepatocytes, such as those treated with phenobarbital.