The molecular mechanisms by which dietary cholesterol is trafficked within cells are poorly understood. Previous work indicates that the NPC1 family of proteins plays an important role in this process, although the precise functions performed by this protein family remain elusive. We have taken a genetic approach to further explore the NPC1 family in the fruit fly Drosophila melanogaster. The Drosophila genome encodes two NPC1 homologs, designated NPC1a and NPC1b, that exhibit 42% and 35% identity to the human NPC1 protein, respectively. Here we describe the results of mutational analysis of the NPC1a gene. The NPC1a gene is ubiquitously expressed, and a null allele of NPC1a confers early larval lethality. The recessive lethal phenotype of NPC1a mutants can be partially rescued on a diet of high cholesterol or one that includes the insect steroid hormone 20-hydroxyecdysone. We also find that expression of NPC1a in the ring gland is sufficient to rescue the lethality associated with the loss of NPC1a and that cholesterol levels in NPC1a mutant larvae are unchanged relative to controls. Our results suggest that NPC1a promotes efficient intracellular trafficking of sterols in many Drosophila tissues including the ring gland where sterols must be delivered to sites of ecdysone synthesis.