Rapid cold hardening (RCH) is an adaptation enabling insects to quickly respond to low temperature, but little is known about the molecular events that trigger this response. In this study of the flesh fly Sarcophaga crassipalpis, we explore a possible role for mitogen-activated protein kinases (MAPKs) in the low temperature signaling that elicits RCH. We report that p38 MAPK from S. crassipalpis, which shows high cDNA sequence homology to p38 MAPKs from other insects and mammals, is rapidly activated at temperatures around 0°C, temperatures that are most effective for inducing RCH. By contrast, low temperature does not activate either extracellular signal-regulated kinase (ERK) or Jun N-terminal kinase (JNK). An increase in phospho-p38 MAPK was observed within 10 min following exposure to 0°C and reached its maximum level in 2 h. When flies were transferred from 0 to 25°C, the level of phospho-p38 MAPK decreased immediately and reached trace levels by 3 h. Nondiapausing flies were much more responsive to p38 MAPK activation than cold-hardy diapausing pupae. Thus, p38 MAPK activation and RCH both show the same narrow ranges of temperature sensitivity, temporal profiles of activation and decay, and developmental specificity. These correlations suggest that p38 MAPK plays a potential role in regulating the induction of RCH. The p38 MAPK response was not dependent upon the brain, as evidenced by high activation in isolated abdomens exposed to low temperature.