Each step in the biogenesis of microRNAs (miRNAs) depends on recognition of a correct substrate and efficient transport or processing of that RNA. Exportin5 (Exp5) and Dicer are proteins that mediate two key steps in this cascade, the nuclear export and cytoplasmic processing of microRNA precursor (pre-miRNAs). Xenopus laevis oocytes, eggs, and embryos constitute convenient experimental systems in which to study the substrate specificity of these proteins because specific RNAs or proteins can be injected directly into different subcellular compartments. We have used the Xenopus system and in vitro processing to define and compare the specificities of Exp5 and Dicer. Although both proteins act on many of the same substrates, we show that they recognize different structure elements of these RNAs. Our studies also revealed several unexpected activities. For example, Exp5 can mediate export of unspliced pre-mRNAs and excised lariat introns if these RNAs contain an aptamer sequence that itself is an Exp5 export substrate. Finally, we demonstrate that maturation of Xenopus oocytes into eggs leads to a large increase in Dicer activity, suggesting that miRNA biogenesis is subject to developmental control.