Phospholipids from homogenate, nuclei, mitochondria, endoplasmic reticulum (ER), plasma membrane (PM), and cytosol of liver and hepatoma 7288CTC (from inbred male BUF rats) were analyzed for their concentrations, fatty acid compositions of individual lipid classes, and levels of octadecenoate positional isomers. The phospholipid concentrations of hepatoma mitochondria and ER were less than 60% of liver values. Sphingomyelin concentrations were elevated dramatically in hepatoma nuclei, mitochondria, and PM. Hepatoma nuclei and mitochondria contained only 25% or less the concentrations of phosphatidylethanolamine (PE) as those of liver, whereas ER, PM, and cytosol fractions of hepatoma contained equal or greater concentration of PE than did the corresponding liver fractions. The fatty acid profiles of the individual lipid classes were somewhat characteristic of liver organelles but not of hepatoma. Lipid classes thought to be located preferentially on the outer bilayer of liver PM contained lower percentages of polyunsaturated fatty acids than did hepatoma PM. For hepatoma generally the lipid classes tended to exhibit a more uniform fatty acid profile among organelles, the polyunsaturated fatty acid percentages were decreased, and the octadecenoate percentages were increased. Octadecenoates isolated from individual lipid classes of organelles contained high levels of cis-vaccenate, in addition to oleate, and some class and organelle specificity was observed in liver. In contrast, hepatoma octadecenoates exhibited little class or organelle specificity, and much higher oleate concentrations were found in PM phosphatidylcholine, PM PE, and ER PE in hepatomas than in liver.