Activated peritoneal macrophages (M phi) from mice injected with Bacilli Calmette-Guérin, trehalose dimycolate, a defined immunostimulant derived from Mycobacterium tuberculosis, or streptococci C74 (St), synthesized two to three times less paf-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in response to a zymosan challenge than did (0.7 +/- 0.2 nmol/mg protein) resident-M phi (R-M phi). To assess at which step the paf-acether biosynthetic pathway was impaired, the content in phospholipid paf-acether precursors was evaluated. The alkyl-acyl-glycerophosphocholine content was comparable in R-M phi (50.4 +/- 18.6 nmol/mg protein) and activated-M phi (40.0 +/- 6.5 to 63.9 +/- 7.7 nmol/mg protein), as well as the lyso paf-acether content (0.85 +/- 0.18 nmol/mg protein for R-M phi vs 0.63 +/- 0.16 to 1.23 +/- 0.21 nmol/mg protein for activated macrophages). The nonlimiting rate of the phospholipid substrate was strengthened by experiments showing that incubation of the various populations with lyso paf-acether did not yield increased amounts of paf-acether. Similarly, incubation of R-M phi or St-M phi with sodium acetate increased paf-acether production to the same extent in both populations, ruling out the acetate substrate deficiency as the cause of the impaired production. The level of acetyltransferase activity, the enzyme that transfers an acetate moiety of acetyl coenzyme A (acetyl-CoA) onto lyso paf-acether, was very low in thioglycolate (TG)-elicited M phi but high in R-M phi and activated ones. In all cases, it was increased by two to three times upon zymosan challenge. This suggested that increased paf-acether catabolism and not impaired anabolism could be responsible for the marked reduced formation noted in activated macrophages. The addition of acetyl-CoA (200 microM) to the various macrophage monolayers restored paf-acether formation by activated cells to R-M phi values but with delayed kinetics as compared with paf-acether formation induced by zymosan. The enhancing effect of acetyl-CoA on paf-acether production was inhibited upon oleyl-CoA addition, suggesting that acetyl-CoA may increase paf-acether production by preventing the reacylation of lyso paf-acether resulting from paf-acether degradation. In conclusion, the paf-acether output in some inflammatory macrophages may be regulated by the level of acetyltransferase activity, because it is observed in TG-M phi. However, we present the first evidence for another mechanism of regulation, most probably related to the deacylation/reacylation of paf-acether precursors and metabolites.