Selected molecular species of rat testicular 1,2-diradyl- sn-glycero-3-phosphocholines and 1,2-diradyl- sn-glycero-3-phosphoethanolamines were quantitated as their diradylglycerobenzoate derivatives, using a recently developed high-performance liquid Chromatographic method. Increased amounts of docosapentaenoic acid were found in glycerophospholipids containing ether moieties compared with the diacyl phospholipids (e.g., docosapentaenoate-containing species comprised more than 80% of the alkylacyl subclass of the ethanolamine phosholipids as opposed to 29.3% of the diacyl subclass). Within 2 h after intratesticular injections of [5,6,8,9,11,12,14,15- 3H]arachidonic acid, the 20:4–20:4 and 18:2–20:4 molecular species of the diacyl subclass of both the choline and ethanolamine glycerophosphatides had the highest specific radioactivities. These unique molecular species (20:4–20:4 and 18:2–20:4) also exhibited the largest percentage decrease in specific radioactivity 24 h after the intratesticular injections of [ 3H]arachidonic acid, which indicates these two species possess a high metabolic turnover. Two of the arachidonate-containing molecular species (18:1–20:4 and 18:0–20:4) in the ethanolamine plasmalogens showed only a small decrease in specific radioactivity, whereas a third species (16:0–20:4) actually had a 44% increase in specific radioactivity 24 h after the intratesticular injections of [ 3H]arachidonate. These data indicate that the 20:4–20:4, 18:2–20:4 and 18:1–20:4 species of phosphatidylcholine and/or phosphatidylethanolamine are most rapidly labeled after administration of [ 3H]arachidonic acid and that they appear to serve as the source of the [ 3H]arachidonate that is ultimately transferred to ethanolamine plasmalogens.