De novo phospholipid biosynthesis was assayed in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) both fully acclimated to 5 or 20 degrees C and undergoing acclimation from one temperature extreme to the other. Incorporation of [14C]choline, [3H]ethanolamine, and [3H]serine into phosphatidyl-choline (PC), phosphatidylethanolamine (PE), or both, was followed to assess metabolic capacity. PE biosynthesis rates exceeded those for PC four- to fivefold. Methylation of PE accounted for 10 (20 degrees C)-17% (5 degrees C) of the synthetic capacity for PC, whereas 6 (20 degrees C-acclimated)-27% (5 degrees C-acclimated) of PE synthesis was derived from phosphatidylserine (PS) decarboxylation. Several factors may contribute to the altered proportions of PE and PC or unsaturated molecular species of phospholipids characteristic of thermally acclimated animals. 1) Activities of choline and ethanolamine phosphotransferase pathways were significantly higher, and decarboxylation activity lower, in 20 degrees C than in 5 degrees C-acclimated trout, resulting in maintained PE synthesis despite a general depression of lipid biosynthesis at cold temperatures. 2) PC biosynthesis depended more on temperature (Q10 = 2.6-3.0) than that of PE (Q10 = 1.8-2.2), causing the ratio of PC/PE synthesis to be positively correlated with temperature. 3) Contribution of methyltransferase pathway to the synthesis of PC was higher at 5 than 20 degrees C. 4) The percentage of ethanolamine incorporation recovered in PC increased threefold in the early stages of warm acclimation. However, not all adjustments in biosynthetic capacity (most notably a 10-fold stimulation of PC synthesis 2 days after transfer of warm-acclimated trout to 5 degrees C) influence membrane lipid composition, implicating other processes in the regulation of this parameter.