From a specialized cDNA library of Giardia lamblia, 20 snoRNA-like RNAs, including 16 box C/D sRNAs and four box H/ACA sRNAs, were first identified. The sRNAs were predicted to guide a total of 11 2'-O-methylation and four pseudo uridylation sites on the G. lamblia rRNAs, respectively. By using primer extension assay, seven methylation sites were precisely mapped in the G. lamblia 16S rRNA, despite its high GC content. All of the sRNA genes locate on the small intergenic regions of the G. lamblia genome and seem to be independently transcribed from their own promoters. Particularly, a cluster composed of GIsR17 and GIsR18 genes is transcribed as a dicistronic precursor, implying a mechanism of endonuclease cleavage for the maturation of the two sRNAs. The systematic identification of the sRNAs in G. lamblia has provided valuable information about the characteristics of the two major families of small guide RNAs in one of the most primitive eukaryotes and would contribute to the understanding of the evolution of small non-messenger RNA genes from prokaryotes to eukaryotes. (C) 2005 Elsevier Inc. All rights reserved.