The effects of altered leader sequences on the secretion and localization of restrictiocin expression in Aspergillus nidulans and Aspergillus niger were investigated. The region encoding the leader sequence of the Aspergillus restrictus restrictocin (res) gene was altered and variants were expressed under the glucoamylase (glaA) promoter in A. nidulans and A. niger. The entire restrictocin leader sequence was replaced by the glaA leader sequence in one variant. In another, the signal sequence of restrictocin was replaced with the glaA signal, leaving a hybrid with the putative restrictocin pro region in place of the glaA pro region. The putative pro region was deleted from the restrictocin leader of a third variant. Toxic effects, such as reduced transcript levels and cellular lysis, were minimal when restrictocin was expressed with the native leader sequence, but became more pronounced as the leader sequence was varied. These toxic effects were inversely proportional to the level of restrictocin secreted. In all transformed strains, restrictocin secretion appeared at the periphery of colonies and was observed to occur at the tips of hyphae. Localization of restrictocin to differentiated structures (conidiophores), as occurs in A. restrictus, was observed only in transformed strains containing the complete restrictocin leader sequence.