Occupancy of specific receptors on neutrophils by adenosine or its analogues diminishes the stimulated release of toxic oxygen metabolites from neutrophils, while paradoxically promoting chemotaxis. We now report evidence that two distinct adenosine receptors are found on neutrophils (presumably the Al and A2 receptors of other cell types). These adenosine receptors modulate chemotaxis and 02 generation, respectively. N6-Cyclopentyladenosine (CPA), a selective A, agonist, promoted neutrophil chemotaxis to the chemoattractant FMLP as well as or better than 5'N-ethylcarboxamidoadenosine (NECA). In contrast, CPA did not inhibit O2 generation stimulated by FMLP. Pertussis toxin completely abolished promotion of chemotaxis by CPA but enhanced inhibition by NECA of 02 generation. Disruption of microtubules by colchicine or vinblastine also abrogated the enhancement by NECA of chemotaxis whereas these agents did not markedly interfere with inhibition by NECA of°2 generation. FMLP receptors, once they have bound ligand, shift to a high affinity state and become associated with the cytoskeleton. NECA significantly increased association of I3HIFMLP with cytoskeletal preparations as it inhibited 2.-Disruption of microtubules did not prevent NECA from increasing association of PHIFMLP with cytoskeletal preparations. Additionally, CPA (Al agonist) did not increase binding of IHIFMLP to the cytoskeleton as well as NECA (A2 agonist). These studies indicate that occupancy of one class of adenosine receptors (Al) promotes chemotaxis by a mechanism requiring intact microtubules and G proteins whereas engagement of a second class of receptors (A2) inhibits 0-generation. Signalling via A2 receptors is independent of microtubules, insensitive to pertussis toxin and is associated with binding of [HjFMLP to cytoskeletal preparations.