Antisense oligonucleotides (ODN), complementary to mRNA of human tumor necrosis factor α (TNFα) and lymphotoxin (LT) were tested for their ability to inhibit TNFs. TNFs production was studied in cell-free systems including wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL). All ODN were effective in WGE at low concentration (0.2 μM) except those targeted to the 3′ region of TNFα mRNA. A short ODN complementary to a common region between TNFα and LT inhibited both TNFs. In contrast, high ODN concentration (50 μM) was needed to inhibit LT mRNA translation in RRL, whereas no clear inhibition of TNFα was observed unless RNase H was added to the translation mixture. ODN effects on TNFs production by stimulated cell line in culture were also investigated. Three ODN — one located in the 5′-untranslated region, one spanning the AUG initiation codon and one downstream of this AUG — were the most effective sequences to decrease TNFα production. Two ODN targeted to the AUG initiation codon of LT were also able to inhibit its production. In conclusion we confirm the role of RNase H in cell free systems, and we found that there is no correlation between ODN efficiency in a cell-free system nor in cell culture. Efficient ODN could be used for in vitro investigation of the role of TNFα and LT in mechanism in which they are involved.