Beginning in early S phase, synchronized Chinese hamster ovary cells were heated for 12 h at 41.5 degrees C, during which time the cells progressed through S, G2, and into mitosis. Upon return to 37 degrees C, some of the interphase cells developed a fragmented nuclear morphology, and the fraction of cells exhibiting this effect reached 40% after entering the first mitosis. About 50% of the mitotic cells accumulated with Colcemid at the second mitosis exhibited premature chromosome condensation (metaphase-associated PCC) in an amount equal to approximately one to two chromosomes per diploid cell. There was also a low frequency of spontaneous S and G2 PCC (0.5 to 1.5% in controls); this distinctly different type of PCC was observed in heated interphase cells at a frequency of 2.0 to 7.0% before the first wave of entry into mitosis. Heated S-phase cells exhibited a chromosome break frequency of about 0.9 breaks per cell when analyzed at the first mitosis. This value agrees well with the observed surviving fraction of 40%, if we assume that one break per cell is a lethal event, and that chromosome damage has a Poisson frequency distribution in the cell population; however, this correlation may not be valid for tumor cell populations. Between the first and second mitoses, an increase in hyperthermia-induced aneuploidy was observed which was apparently due to unequal division, since mitotic cells containing PCC were excluded from the chromosome number frequency analyses. Although this induction of aneuploidy was observed independently of PCC, we conclude that abnormal division is necessary for both effects, and both PCC and unequal division result in an increase in aneuploidy. Previous studies using transmission electron microscopy demonstrated a precocious reformation of the nuclear membrane around metaphase chromosomes in Chinese hamster ovary cells heated at this temperature (R. A. Coss et al., Cancer Res., 39: 1911-1918, 1979). We suggest that such heat-induced changes in nuclear envelope formation lead to nuclear fragmentation and ultimately result in metaphase-associated PCC.