In animals and protozoa, gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular RNAs, a phenomenon known as RNA interference (RNAi). In vitro and in vivo dsRNA is processed by a nuclease to produce 21–25-nt small interfering RNAs (siRNAs) that guide target RNA degradation. Here we show that activation of RNAi in Trypanosoma brucei by expression or electroporation of actin dsRNA results in production of actin siRNAs and that 10% of these RNAs sediment as high-molecular-weight complexes at 100,000 × g. To characterize actin siRNAs, we established a cloning and enrichment strategy starting from 20–30 nt RNAs isolated from high-speed pellet and supernatant fractions. Sequence analysis revealed that actin siRNAs are 24–26 nt long and their distribution relative to actin dsRNA was similar in the two fractions. By sequencing over 1,300 fragments derived from the high-speed pellet fraction RNA, we found abundant 24–26-nt-long fragments homologous to the ubiquitous retroposon INGI and the site-specific retroposon SLACS. Northern hybridization with strand-specific probes confirmed that retroposon-derived 24–26-nt RNAs are present in both supernatant and high-speed pellet fractions and that they are constitutively expressed. We speculate that RNAi in trypanosomes serves a housekeeping function and is likely to be involved in silencing retroposon transcripts.