We have studied the metabolism of selected diacyl molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and diacylglycerol (DG) from rat rod outer segments (ROS). Rats were injected intravitreally with [2-3H]glycerol. At 1, 2, 3, 4 and 6 days post-injection, ROS phospholipids and DG were isolated by two-dimensional thin-layer chromatography (TLC), derivatized, and fractionated into molecular species by high-performance liquid chromatography (HPLC). Selected molecular species were quantitated and counted for radioactivity. We found the following. In PC and PE, the specific activities of 22:6-22:6, 18:1-22:6 and 16:0-22:6 were highest at day 1 and then decreased in a nearly exponential manner. In contrast, the specific activities of 18:0-22:6 and 18:0-20:4 were substantially lower than these three molecular species and changed little over the 6-day period. In PS, the specific activities of 22:6-22:6, 18:0-22:6 and 18:1-22:6 were similar and did not reach their maximum until the 3rd or 4th days. In PC, the specific activities of the five molecular species examined were two to three times higher at day 1 than the same species in PE and PS. In PI and DG, the major molecular species were 16:0-20:4 and 18:0-20:4. The specific activities of these two molecular species at day 1 were about ten times higher than those of 20:4-containing species in PE and PC, and showed the most rapid turnover of any of the molecular species examined in this study.(ABSTRACT TRUNCATED AT 250 WORDS)