The relationship of polyamine accumulation and semiconservative DNA replication was studied in synchronous Chinese hamster ovary cultures, progressing through the cell cycle either normally at 37 degrees or after hyperthermic exposure (43 degrees for 1 hr) during G1 or S phase. In control cultures, intracellular polyamine levels decreased as cells divided and then reaccumulated as cells exited G1 and proceeded through the S and G2 phases. Immediately after cultures were exposed to 43 degrees heat for 1 hr in G1 phase, intracellular levels of spermidine and spermine were reduced compared to controls. Coordinate with the depletion of the intracellular levels of these polyamines following exposure at 43 degrees, extracellular levels of spermidine and spermine were increased. The ratio of intracellular to extracellular amounts of both these polyamines changed from 1 to 1.5 to approximately 0.2 to 0.3 after hyperthermic exposure. These cultures exposed to 43 degrees heat during G1 initially showed depressed levels of replicated DNA, but near-control rates of DNA replication were atained in a temporally related manner with the reaccumulation of intracellular spermidine and spermine levels. When cultures were exposed to 43 degrees heat in S phase, intracellular amounts of spermidine and spermine were again reduced and increased extracellular levels of these polyamines were observed. In these S-phase-treated cultures, cells were able to continue replicating their DNA but at a much reduced rate compared to controls. These results and other show that: (a) exposure of cells at 43 degrees causes a depletion of intracellular levels of spermidine and spermine, suggesting that an immediate aspect of thermal damage is a membrane defect that markedly affects the transport of these molecules across cell membranes; (b) exposure of either G1- or S-phase cultures to 43 degrees heat caused a depression of bulk DNA-synthetic rates resulting in a prolongation of S phase, and (c) the intracellular reaccumulation of spermidine and spermine following exposure of G1 cells to a 43 degrees heat shock is temporally related to the recovery of near-normal DNA synthetic rates in these cells.