Hairpin-structured (hp) RNA has been widely used to induce RNA interference (RNAi) in plants and animals, and an in vivo expression system for hpRNA is important for large-scale RNAi applications. Bacterial expression systems have so far been developed for in vivo expression of hpRNA or double-stranded (ds) RNA, but the structure of the resulting RNAi molecules has remained unclear. Here we report that long hpRNAs expressed in the bacteria Escherichia coli and Sinorhizobium meliloti were largely processed into shorter dsRNA fragments with no or few full-length molecules being present. A loss-of-function mutation in the dsRNA-processing enzyme RNase III, in the widely used E. coli HT115 strain, did not prevent the processing of hpRNA. Consistent with previous observations in plants, the loop sequence of long hpRNA expressed in Agrobacterium-infiltrated Nicotiana benthamiana leaves was excised, leaving no detectable levels of full-length hpRNA molecule. In contrast to bacteria and plants, long hpRNAs expressed in the budding yeast Saccharomyces cerevisiae accumulated as intact, full-length molecules. RNA extracted from hpRNA-expressing yeast cells was shown to be capable of inducing RNAi against a β-glucuronidase (GUS) reporter gene in tobacco leaves when applied topically on leaf surfaces. Our results indicate that yeast can potentially be used to express full-length hpRNA molecules for RNAi and perhaps other structured RNAs that are important in biological applications.