MicroRNAs (miRNAs) are small, noncoding regulatory RNA molecules that bind to 3' untranslated regions (UTRs) of mRNAs to either prevent their translation or induce their degradation. Previously identified in a variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by viruses. The human gammaherpesvirus Epstein-Barr virus has been shown to encode miRNAs, which potentially regulate both viral and cellular genes. To determine whether Kaposi's sarcoma-associated herpesvirus (KSHV) encodes miRNAs, we cloned small RNAs from KSHV-positive primary effusion lymphoma-derived cells and endothelial cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align with KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latency-associated region of KSHV. These data suggest that viral and host cellular gene expression may be regulated by miRNAs during both latent and lytic KSHV replication.