The effect of prolonged light halothane anesthesia (0.8%) on the proliferation rate of different mouse tissues was investigated, using [5-125I]5-iodo-2-deoxyuridine uptake into DNA as the test parameter. It was found that DNA synthesis in spleen, femoral bone marrow, and, occasionally, the small intestine was significantly depressed after exposure for 24 hr to halothane in vivo. The time course of DNA synthesis inhibition was then investigated by utilizing a shorter (6-hr) exposure time. This period was found to be insufficient to cause DNA synthesis inhibition in any of test tissues. Because anesthesia was found to be associated with hypothermia at normal room temperatures, it was established that the inhibition of DNA synthesis was not due to cooling of the mice under anesthesia by demonstrating that inhibition in sensitive tissues occurred at warmer temperatures as well. To examine the specificity of this finding, the DNA synthesis rate of cells in other normal tissues, e.g., skin and muscle, and in s.c.-growing tumor cells of a mouse mammary carcinoma, L1210 leukemia, and a first transplant AKR lymphoma were examined. In none were responses noted with 24 hr of halothane exposure. However, halothane was found to inhibit DNA synthesis in regenerating marrow. Finally, it was found that after significant exposure to halothane, complete recovery was seen in the spleen after 24 hr, whereas femur DNA synthesis was still depressed by 20% at the same time.