Fish antifreeze proteins (AFPs) have been reported by some researchers to prolong the viability of tissues, organs, and embryos under hypothermic conditions, while others have observed no such effect or even AFP-mediated cryotoxicity. We examined the influence of Type III AFP from Atlantic wolffish on cold tolerance in a whole animal model system, transgenic Drosophila. The activity of the AFP, transgenically expressed under the transcriptional control of the female-specific yp1 and yp2 promoters and secreted into fly hemolymph, was confirmed through thermal hysteresis and differential scanning calorimetry measurements as well as through observations of ice crystal morphology. In cold exposure trials, at 0 degrees C and at -7 degrees C, transgenic adult flies of both sexes exhibited greater survival than nontransgenic controls even though the antifreeze was only produced in females. We attribute these observations to the expression of the xanthine dehydrogenase marker gene used to identify transgenics, rather than the production of AFP. Type III AFP therefore appears unable to enhance survival of adult Drosophila under hypothermic conditions.