Drosophila melanogaster, a freeze intolerant and cold shock sensitive insect, was transformed with the hyperactive insect antifreeze protein gene (AFP) from the spruce budworm, Choristoneura fumiferana. Transformation P-element constructs (pCasper) were made with CfAFP 337 isoform DNA using a strong constitutive promoter, Actin 5c. This is the first report of insect AFP used to transform another insect. Properly folded active insect AFP was only detected when signal sequences were used to target proteins to the endoplasmic reticulum for secretion into the hemolymph. The 18 residue Drosophila binding protein signal sequence (BiP) constructs resulted in transformed fly lines with significantly higher AFP expression in hemolymph than when the native C. fumiferana AFP signal sequence was used. The resultant transgene fly lines have the highest levels of thermal hysteresis, 0.8 degrees C, seen for any engineered Drosophila. Despite the high level of expression, even higher than some overwintering fish with natural levels of endogenous AFP, the transformants did not display any cold shock resistance compared to controls or low AFP expressing lines. These results indicate that insect AFP alone cannot protect Drosophila from cold shock and may not be useful for Drosophila cryopreservation.