Chemotaxis by a macrophage cell line, RAW264, is specifically inhibited by the intracellular accumulation of 3-deazaadenosylhomocysteine (3-deaza-AdoHcy). Intracellular accumulation of 3-deaza-AdoHcy is the result of incubation of the cells with 3-deazaadenosine, a compound that can function both as a substrate and as an inhibitor of adenosylhomocysteine (AdoHcy) hydrolase. Accumulation of AdoHcy per se, brought about in chemotactic cell lines by incubation with either 3-deazaadenosine or with 3-deazaaristeromycin, does not affect chemotaxis. The specific role of 3-deazaadenosine as an inhibitor of macrophage cell line chemotaxis is supported by the following findings. Another macrophage cell line, RAW309CR, is resistant to the inhibition of chemotaxis by 3-deazaadenosine, and the resistance can be ascribed to the failure to accumulate 3-deaza-AdoHcy in this cell line. It is noteworthy that both RAW264 and RAW309CR accumulate similar amounts of AdoHcy after incubation with 3-deazaadenosine. The difference in the accumulation of 3-deaza-AdoHcy in RAW264 and RAW309CR is explained by the finding that sonicates of RAW264 rapidly form 3-deaza-AdoHcy but sonicates of RAW309CR do not. Both hydrolysis of AdoHcy and the inhibition of the hydrolysis of AdoHcy by 3-deazaadenosine are the same in sonicates of the two macrophage cell lines.