Two hammerhead ribozymes flanked by Giardia lamblia alcohol dehydrogenase E (ADHE) antisense RNA fragments, ARzS and ARzL, were designed, synthesized and found capable of cleaving an ADHE mRNA fragment at the anticipated position in vitro. The ribozymes were then electroporated into Giardia trophozoites and expressed via the giardiavirus-mediated RNA expression system. Expression of the ribozyme with two short antisense arms, ARzS, was stabilized under puromycin selection and demonstrated a 33% reduction in ADHE mRNA and 25% decrease in NAD+-dependent ADH activity in the transfectants. Expression of ARzL, the ribozyme with two long antisense arms, cannot be enriched under puromycin without killing the transfected cells, probably due to excessive depletion of ADHE. Without the drug selection, however, transient expression of ARzL 20-40 h after electroporation resulted in an 83.7% loss of ADHE mRNA and an 84.5% reduction in ADH activity in the transfected cells. When the ribozyme moiety was removed from ARzL, the latter retained some of its in vivo activity of lowering ADHE mRNA and ADH activity, suggesting that inhibition of ADHE gene expression in Giardia can be accomplished by the antisense RNA alone, albeit less efficiently. The ADHE deficient transfectant demonstrated relatively poorer anaerobic growth but grew more vigorously than the wild type under aerobic conditions, suggesting that the role of ADHE in providing NAD+ through anaerobic reduction of acetyl-CoA to ethanol could be replaced by a yet unidentified aerobic enzyme(s) in Giardia. The close association consistently observed between the levels of ADHE mRNA and ADH activity in transfected Giardia cells suggests that ADHE could be the only functional alcohol dehydrogenase in Giaradia. One other Giardia gene encoding a putative Class III ADH, GIADH3, was identified and cloned, but no Class III ADH activity could be detected in Giardia by the conventional enzyme assays. This gene is thus probably unexpressed in Giardia trophozoite.