For many years, non-freezing cold shock injury has been associated with damage to the cell membrane. In this study, we enhanced membrane cholesterol levels of Drosophila melanogaster by raising larvae on a cholesterol-augmented diet. Diet augmentation significantly increased the amount of cholesterol in the cell membranes of the adult flies (1.57+/-0.17 nmol per mg vs. 0.93+/-0.11 nmol per mg). Flies on the cholesterol-augmented diet exhibited a greater intrinsic cold tolerance: this group had a higher survival rate after a 2-h cold shock of -5 degree C than did the control group (71.0+/-6.6 percent vs 36.0+/-8.1 percent). Cholesterol-augmented flies also had a significantly greater capacity to rapidly cold-harden to -7 degree C (36.7+/-4.4 percent) compared to flies on a control diet (20.0+/-2.9 percent). These results suggest a mechanistic link between protection from chilling or cold shock injury and modifications to the cellular membrane.