Entomopathogenic nematodes are excellent organisms for dissecting the molecular basis of parasitism and probing the insect innate immune system. The nematode parasite Steinernema carpocapsae is a potent pathogen of insects that has emerged recently as a model for parasitic infection and anti-nematode immune signaling and response. The nematodes are mutualistically associated with the bacteria Xenorhabdus nematophila, which are also pathogenic to insects. Separation of nematodes from their associated bacteria facilitates mechanistic studies focusing on the impact of the parasites without considering the contribution of their bacterial partners. An important aspect in insect infection experiments with entomopathogenic nematodes includes the storage duration of the parasites. Here we have infected larvae of the model insect Drosophila melanogaster with S. carpocapsae nematodes that had been stored for 3 wk or 3 mo. Survival data consistently revealed that infective juveniles with prolonged storage exhibit substantially increased virulence toward D. melanogaster larvae compared with those that had been stored for a shorter time, and the presence of mutualistic X. nematophila in the nematodes does not influence this result. Although the basis for this effect is currently unknown, these surprising findings indicate that prolonged nematode storage can markedly alter virulence. This is significant knowledge that should be taken into account in functional assays involving infection with parasitic nematodes. Future efforts will focus on the identification and characterization of the factors that might determine the interrelationship between prolonged storage and virulence in nematode parasites.