The effect of 5'-methylthioadenosine (MTA) on human natural killer (NK) cell activity was examined and compared with the effect of 3-deazaadenosine (c3-ado) and periodate-oxidized adenosine (ado-ox). MTA inhibited NK cell activity in concentrations above 30 microM, but in concentrations below 10 microM a slight enhancing effect was often observed. C3-ado and ado-ox were 10 and 3 times more potent, respectively, as inhibitory agents and did not increase NK cell activity in low concentrations. The inhibitory effect of c3-ado was unaffected by preincubation of the cells but was enhanced by the addition of L-homocysteine. In concentrations that caused inhibition of NK cell activity all three agents caused a fall in the methylation index (AdoMet/AdoHcy) but no or an inconsistent effect on the level of cyclic AMP. An increase in the level of AdoHcy was observed already after 1 h of incubation but was more pronounced after 4 h of preincubation with the adenosine derivatives. The inhibition of cytotoxicity was mainly on their initiation of lysis, with a smaller effect on target cell binding. Antibody-dependent cellular cytotoxicity and lectin-dependent cellular cytotoxicity appeared to be less sensitive to inhibition by c3-ado. Our results show that several adenosine analogues inhibit NK-cell-mediated cytotoxicity in parallel with a decreased methylation index. The results suggest that a methylation step is critical in lymphocyte-mediated cytotoxicity and that NK cell activity is more sensitive to inhibition of this step than antibody- or lectin-dependent cytoxicity.