We have investigated the hypothesis that adenosine, a purine nucleoside produced within hypoxic regions of solid tumors, may interfere with the recognition of tumor cells by cytolytic effector cells of the immune system. We measured the adhesion of murine spleen-derived anti-CD3-activated killer (AK) lymphocytes to syngeneic MCA-38 colon adenocarcinoma cells in a model system. Adenosine, in the presence of the adenosine deaminase inhibitor coformycin to prevent the breakdown of adenosine, inhibited adhesion by up to 60%. The inhibitory effect of adenosine was exerted on the AK cells and not on the MCA-38 targets. The response to adenosine was generated at the cell surface, since the inhibition of adhesion was not abrogated by S-(4-nitrobenzyl)-6-thioinosine or dipyridamole, which block adenosine uptake. The inhibition of adhesion due to adenosine was not blocked by either the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine. This suggested that a non-A1, A2 receptor might be involved. The relative order of potencies of adenosine and common analogues was: 5'-N-ethylcarboxamidoadenosine = adenosine = (R)-phenylisopropyladenosine > N6-cyclopentyladenosine > 2-chloro-N6-cyclopentyladenosine = 2-p-(2- carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine. This agonist potency profile was again inconsistent with either the A1 or the A2 receptor subtype but indicated that the recently described A3 receptor subtype might be responsible for the inhibition of adhesion. Consistent with this suggestion, aminophenylethyladenosine, an adenosine analogue that binds with high affinity to A3 receptors, inhibited the adhesion of AK cells to MCA-38 tumor cells with high potency (50% inhibitory concentration approximately 1 nM). Adenosine, therefore, interferes with the AK cell recognition of colorectal tumor targets by acting through an A3 receptor on the effector cells. We suggest that this mechanism of immunosuppression, secondary to tissue hypoxia, may be important in the resistance of colorectal and other solid cancers to immunotherapy.