Larvae of Sarcophaga crassipalpis destined for pupal diapause (light:dark 12:12, 20°C) contain nearly twice as much lipid and twice the haemolymph protein concentration as larvae that will not enter diapause (light:dark 15:9, 20°C). This conspicuous difference in metabolic reserves provides the earliest indication of the developmental fate of the larva. Lipid reserves are utilized rapidly during the first half of diapause and then remain stable until adult eclosion. In contrast, residual dry weight changes very little early in diapause but drops sharply late in diapause, thus implying a transition from lipid utilization to protein or carbohydrate utilization in mid-diapause. We suggest that this metabolic transition marks the end of the “fixed latency period”: pupae readily respond to environmental or hormonal stimulation after this point. Diapause-destined larvae did not accumulate more glycogen than nondiapause-destined larvae, but an 80% decrease in glycogen at the onset of diapause and its elevation at the end of diapause suggests the utilization of glycerol or related compounds as cryoprotectants during diapause. Profiles of water content are very similar in short-day and long-day flies, thus suggesting that dehydration is not a mechanism exploited by the flesh fly to achieve cold hardiness. Adult flies that have experienced pupal diapause emerge from the puparium with lipid, glycogen, and water content nearly identical to flies that have not experienced diapause, but the residual dry weight is much lower. The severe depletion of protein may account for the reduced fecundity of flies that have experienced diapause.