A gene encoding a 68.5-kDa trophozoite surface protein (TSP11) of the Australian Giardia intestinalis (syn. G. lamblia) isolate, Ad-1, has been cloned from a genomic expression library screened with an antiserum specific for 3 major surface antigens. Sequence analysis of two overlapping genomic fragments identified a single open reading frame that contained no introns and predicted a cysteine-rich, 667-residue polypeptide with features common to other trophozoite surface proteins. These include the presence of 27 copies of the 4-amino acid Cys-X-X-Cys motif, an N-terminal signal sequence and a highly conserved, hydrophobic C-terminal segment. Transcripts from the tsp11 gene were detected as a single band on Northern blots using total RNA extracted from Ad-1 trophozoites. Primer extension analysis indicated that the mRNA has a 5' untranslated region of only 5 nt, similar to the very short (1-6 nt) leader sequences reported for other Giardia mRNAs. A large portion of the promoter distal segment of tsp11 has homology with tsa417, a gene encoding a 72.5-kDa trophozoite surface antigen of the Afghanistan-derived G. intestinalis isolate, WB .