In most studies of fatty acid and lipid synthesis in plants there has been poor incorporation of radioactive label from acetate into linoleic (18:2) and linolenic (18:3) acids. Consequently the amounts of these fatty acids found in the galactolipids in such studies are much less than their observed endogenous levels. In the present study incorporation of H
14
CO
3
-
and (1
14
C) acetate into lipids of barley protoplasts was examined. CO
2
-dependent O
2
evolution rates of the protoplasts were around 180 µmol O
2
/h/mg Chl and intactness was also ascertained by phase contrast microscopy. Incubating proto- plasts with 1mM H
14
CO
3
-
or 50 µM (1-
14
C) acetate resulted in 146.2 and 17 nmol/mg Chl being incorporated into lipids respectively after 1 hour. A concentration of 10 mM was optimal for HCO
3
-
incorporation and up to 580 nmol/mg Chl was incorporated into lipids at the end of 1 hour. Mg
++i
ions used at 2 mM had little effect on HCO
3
-
incorporation while PP
i
appeared to be slightly inhibitory. Acetate assimilation and its incorporation into lipids was markedly affected by pH and pH 5.8 was chosen for the assay medium. In 20 hour incubations 162 nmol acetate/mg Chl was incorporated. About 33% of label from acetate was found in each of palmitic (16:0) and oleic (18:1) acids with less than 9% in each of stearic (18:0), linoleic and linolenic acids. There was little or no incorporation of acetate into DGDG and less than 10% into each of PG, MGDG, PE and U (unknown lipid). Incorporation into PC after 2 1/2 hours was 36.8% then decreased to 8.9%. Acetate incorporation was most significant into U
SF
(another unknown lipid), being 73.4%. Although acetate was incorporated into a range of glycerolipids, incorporation into constituent 18:2 and 18:3 of these lipids was not significant.
