The nuclear transport of classical nuclear localization signal (cNLS)-containing proteins is mediated by the cNLS receptor importin alpha. The conventional importin alpha gene family in metazoan animals is composed of three clades that are conserved between flies and mammals and are referred to here as alpha1, alpha2, and alpha3. In contrast, plants and fungi contain only alpha1 genes. In this study we report that Drosophila importin alpha3 is required for the development of both larval and adult tissues. Importin alpha3 mutant flies die around the transition from first to second instar larvae, and homozygous importin alpha3 mutant eyes are defective. The transition to second instar larvae was rescued with importin alpha1, alpha2, or alpha3 transgenes, indicating that Importin alpha3 is normally required at this stage for an activity shared by all three importin alpha's. In contrast, an alpha3-specific biochemical activity(s) of Importin alpha3 is probably required for development to adults and photoreceptor cell development, since only an importin alpha3 transgene rescued these processes. These results are consistent with the view that the importin alpha's have both overlapping and distinct functions and that their role in animal development involves the spatial and temporal control of their expression.