There are two human arylamine N-acetyltransferases (NAT1 and NAT2) that have evolved separately and differ in their substrate specificity and tissue localization. In addition to its acetyltransferase activity, NAT1 can hydrolyze acetyl coenzyme A to coenzyme A in the presence of folate. Here, we show that NAT1 is rapidly inactivated at temperatures above 39 °C whereas NAT2 is more stable. NAT1 acetyltransferase activity is also rapidly lost in whole cells at a rate similar to that of recombinant protein, suggesting it is not protected by intracellular chaperones. By contrast, the hydrolase activity of NAT1 is resistant to heat-induced inactivation, in part because folate stabilizes the protein. Heat generated by mitochondria following the dissipation of the inner membrane potential was sufficient to inactivate NAT1 in whole cells. Within the physiological range of core body temperatures (36.5-37.5 °C), NAT1 acetyltransferase activity decreased by 30% while hydrolase activity increased by >50%. This study demonstrates the thermal regulation of NAT1, but not NAT2, and suggests that NAT1 may switch between an acetyltransferase and a hydrolase within a narrow temperature range in the presence of folate.