With the ultimate aim of developing an effective antiviral strategy against HIV‐1, a mono‐DNA enzyme possessing the 10–23 catalytic motif [Santoro and Joyce (1997) Proc. Natl. Acad. Sci. USA 94, 4264–4266] was synthesized against the HIV‐1 envelope gene. We tested the in vitro cleavage efficiency of the 178 bp long truncated HIV‐1 Env transcript by DNA enzyme 6339. Protein independent and Mg²⁺ dependent specific cleavage products were obtained. As soon as 5 min after mixing equimolar concentrations of DNA enzyme and substrate RNA, more than 50% cleavage was observed which increased steadily over a period of 4 h. Very little cleavage was obtained at 1 mM MgCl₂ concentration which improved significantly when the concentration of MgCl₂ was increased up to 20 mM. Specific inhibition of cell membrane fusion caused by the interaction of gp160 and CD4 in HeLa cells was observed when the above DNA enzyme was used. Thus, these chemically synthesized DNA enzymes could prove to be very useful for in vivo application.