Frost (Fst) is a candidate gene associated with the response to cold in Drosophila melanogaster because Fst mRNA accumulation increases during recovery from low temperature exposure. We investigated the contribution of Fst expression to chill-coma recovery time, acute cold tolerance and rapid cold hardening (RCH) in adult D. melanogaster by knocking down Fst mRNA expression using GAL4/UAS-mediated RNA interference. In this experiment, four UAS-Fst and one tubulin-GAL4 lines were used. We predicted that if Fst is essential for cold tolerance phenotypes, flies with low Fst mRNA levels should be less cold tolerant than flies with normal levels of cold-induced Fst mRNA. Cold-induced Fst abundance and recovery time from chill-coma were not negatively correlated in male or female flies. Survival of 2 h exposures to sub-zero temperatures in Fst knockdown lines was not lower than that in a control line. Moreover, a low temperature pretreatment increased survival of severe cold exposure in flies regardless of Fst abundance level during recovery from cold stress, suggesting that Fst expression is not essential for RCH. Thus, cold-induced Fst accumulation is not essential for cold tolerance measured as chill-coma recovery time, survival to acute cold stress and RCH response in adult D. melanogaster.