Fischer rats bearing s.c. implants of TCT-4909 bladder tumor were treated either with ultrasonic hyperthermia (US) (44.2 degrees, 20 min) or either of two alkylating agents, thiotepa or Cytoxan (CTX) or in combinations of chemotherapeutic agent and heat at specific time intervals. Applying US 20 hr before either agent (thiotepa, 2 mg/kg i.p.; or CTX, 50 mg/kg i.p.) had a less than additive effect upon tumor growth. CTX, administered 20 hr before US, resulted in a significantly increased tumor volume-doubling time compared to CTX only. This was not true for thiotepa. With both agents, a synergistic effect was obtained when US and the agent were applied within 1 hr of each other, but the maximum was observed when the US had been applied 30 min before injection of agent. The intratumor temperature had decayed to normal at the time of injection. Radiolabeled alkylating agents injected at different times after US showed decreased uptake of label up to 20 hr after heating. Tritiated thymidine uptake was also reduced over the same period. Nuclear morphometry indicated increased nuclear condensation in parallel with the reduced uptakes described above. The data suggest that the synergism was not due to increased uptake of agent into heated tissue nor to the direct activation of alkylating activity by heat. It was demonstrated that heat had a rapid and marked inhibitory action upon DNA synthesis. This could have augmented the delayed but prolonged DNA inhibition caused by the alkylating agents to produce a synergistic effect. The apparent prolongation of the growth-inhibitory effect of CTX or thiotepa by heat may be due to the thermal inhibition of the enzymes responsible for the recovery of DNA after alkylation. The precise mechanism for the synergy may vary with the agent, the dose, the equilibrium temperature and its dwell time, and the interval between modalities. The influence of each of these parameters will require further investigation.