The fatty acid content and composition of hepatic microsomes of separated smooth and rough components and of isolated phosphatidylcholine and phosphatidylethanolamine fractions were studied in male albino rats treated with phenobarbital or carbon tetrachloride. Both test compounds significantly altered the fatty acid composition of the endoplasmic reticulum. The total amount was significantly raised by phenobarbital and reduced by carbon tetrachloride. Phenobarbital enhanced palmitic, stearic, arachidic, palmitoleic, linoleic, eicosenoic, eicosadienoic, eicosatrienoic, eicosapentenoic, docosatrienoic, and docosahexenoic acids. Carbon tetrachloride diminished all these, excluding palmitic and palmitoleic acids. The fatty acid content of rough microsomes was significantly increased by phenobarbital and decreased by carbon tetrachloride, while in smooth microsomes fatty acids were raised by phenobarbital but mainly unaffected by carbon tetrachloride. In microsomal phosphatidylcholine fractions, phenobarbital significantly elevated oleic, linoleic, eicosatrienoic, arachidonic, eicosapentenoic, docosapentenoic, and docosahexenoic acids, whereas all these were significantly reduced with carbon tetrachloride. In phosphatidylethanolamine fractions, phenobarbital increased palmitoleic, oleic, linoleic, and arachidonic acids; carbon tetrachloride elicited opposite effects on these acids. Phenobarbital increased and carbon tetrachloride reduced the fatty acid content in the phosphatidylcholine fraction of rough membranes. Opposite effects were seen in oleic, linoleic, arachidonic, and eicosapentenoic acids. Both test compounds brought about similar changes in the fatty acid composition of the phosphatidylethanolamine fractions of rough microsomes. In smooth microsomes, phosphatidylcholine fatty acids were significantly enhanced by phenobarbital and reduced by carbon tetrachloride. The fatty acid content of phosphatidylethanolamine was increased by phenobarbital, mainly manifesting in palmitoleic, oleic, linoleic, arachidonic, docosapentenoic, and docosahexenoic acids. Carbon tetrachloride elicited no major change in this fraction. Phenobarbital increased the production of unsaturated fatty acids, whereas carbon tetrachloride elevated the relative amount of saturated fatty acids. The saturated/unsaturated fatty acids ratio was reduced by phenobarbital and increased by carbon tetrachloride, and thus may indicate a selective difference between an inducer and hepatotoxin on fatty acid synthesis of the hepatic endoplasmic reticulum.