We examined the effects of extracellular ATP on the proliferation and synthesis of DNA by guinea pig alveolar macrophages (AM). AM proliferated spontaneously in vitro, their number doubling in 72 h. Such proliferation was completely inhibited by adding 1 mM ATP to the culture. The inhibition was dose dependent. ATP also suppressed the spontaneous synthesis of DNA by AM. The inhibitory effect of ATP was not related to cell damage, as the viability and the superoxide anion-generating activity of these cells were unaffected by treatment with ATP for 24 h. The order of potency of the adenosine nucleotides (ATP > ADP > AMP) reflected the character of the P2 purinoceptor. Theophylline inhibited the effect of ATP on the synthesis of DNA by AM to a level produced by the nonhydrolyzable analogue, ATP gamma S, but did not influence the effect of ATP gamma S. These data suggest that the effect of ATP on the synthesis of DNA was exerted mainly via the P2 purinoceptor (82.0%) and to a lesser extent via the P1 purinoceptor (12.6%). We found that small molecules in the lavage fluid inhibited the synthesis of DNA by AM. Thus, the extracellular ATP present in the alveolar lining fluid may participate in controlling the proliferation of AM.