Rat peritoneal macrophages were cultured with a specific and potent phospholipase A2 activator A 23187, with 1-stearoyl-2-[3H]arachidonoyl-sn-GPC as source of [3H] arachidonic acid, and with a dialkyl-GPC, at 2, 10 or 20 microM. Four dialkyl-GPCs were prepared by chemical synthesis. Position 2 of rac-glycerol was alkylated with an alkane chain of 8 carbons and position 1 was alkylated with various alkane chains (8, 10, 12, or 16 carbons). [3H] arachidonic acid was split, then recovered with cell nonesterified fatty acids and nonphosphorous glycerolipids after endocellular phospholipase A2 activity. It was also recovered with fatty acids and eicosanoids isolated from culture medium. Inhibition of fatty acid release and eicosanoid synthesis depended on mixed chain dialkyl-GPC structures. The highest inhibitory effect on arachidonic acid release was reached with 1-decyl-2octyl-GPC and was practically as high in culture medium (IC50 at 5 microM) as in cells (IC50 at 4 microM). 1,2-di-octyl-GPC and 1-dodecyl-2-octyl-GPC had weaker inhibitory effects (but higher in culture medium than in cells). The asymmetrical 1-hexadecyl-2-octyl-GPC poorly affected enzyme activity.