The Iroquois (Irx) genes encode evolutionary conserved homeoproteins. We report that Xenopus genes Irx1 and Irx3 are expressed and required during different stages of Xenopus pronephros development. They are initially expressed during mid-neurulation in domains extending over most of the prospective pronephric territory. Expression onset takes place after kidney anlage specification, but before pronephric organogenesis occurs. Later, during nephron segmentation, expression becomes restricted to the intermediate tubule region of the proximal-distal axis. Loss- and gain-of-function analyses, performed with specific morpholinos and inducible wild-type and dominant-negative constructs, reveal a dual requirement for Irx1 and Irx3 during pronephros development. During neurula stages, these genes maintain the specification of the pronephric territory and define its size. This seems to occur, at least in part, through positive regulation of Bmp signalling. Subsequently, Irx genes are required for proper formation of the intermediate tubule. Finally, we find that retinoic acid signalling activates both Irx1 and Irx3 genes in the pronephros.