MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We describe here a straightforward method to generate retroviral miRNA sponge constructs using a single directional ligation reaction. This approach allows generation of sponges containing more than 20 miRNA binding sites. We provide a basis for the design of the sponge constructs with respect to the sequence of the miRNA binding site and the sequences flanking the miRNA binding sites. In-silico validation approaches are presented to test the predicted efficiencies of the sponges in comparison to known target genes. In addition, we describe in vitro validation experiments to confirm the effectiveness of the miRNA sponges. Finally, we describe how the here described procedure can be adapted to easily generate sponges that target multiple miRNAs simultaneously. In summary, our approach allows rapid generation of single or combination miRNA sponges that can be used for long-term miRNA loss-of-function studies. (C) 2012 Elsevier Inc. All rights reserved.