Phosphorothioate (PT) oligonucleotides are designed as specific agents for antisense therapy although they have been reported to exert non-specific immunomodulatory effects. To elucidate further their actions, the effect of PT deoxyguanosine oligomers (S-oligo(dG)) on in vitro cytokine production by mouse splenocytes was studied. S-oligo(dG)20 inhibited production of INF gamma induced by Con A, E. coli DNA or the combination of PMA and calcium ionophore A23187. The diester analogue was inactive, and of PT homo-oligomers tested, S-oligo(dG)20 was the most active. PT compounds with as few as 5 dG residues could also block INF gamma production. These results indicate that base composition and length, as well as the PT backbone, contribute to the inhibition of INF gamma production and extend the range of immunomodulatory effects of PT compounds.