Normal and hepatoma bearing rats were fed a low level of methyl 2-hexadecynoate in a low fat diet for one month. The effect of the acetylenic acid on lipid metabolism as derived from mass analysis of lipid classes, fatty acids and positional monoene isomers isolated from the major lipid classes of liver and hepatoma has been assessed. Methyl 2-hexadecynoate caused a 25% decrease in body weight and the appearance of essential fatty acid deficiency symptoms within one week. Non-tumor-bearing animals contained a seven-fold increase in all neutral lipid classes, except cholesterol, while host animals did not contain fatty livers. The apparent protective effect of the host animal by the hepatoma also resulted in only marginal changes in the fatty acid and positional monoene isomers from host liver and hepatoma lipids. In contrast to host liver and hepatoma, methyl 2-hexadecynoate caused a massive accumulation of palmitate and hexadecenoates with a concomitant decrease in stearate and octadecenoates in most of the lipid classes from non-tumor-bearing animals. These changes were accompanied by a shift from the higher molecular weight triglycerides to lower molecular weights corresponding to carbon number 48. The high concentrations of hexadecenoates consisted predominantly of the delta 9 isomer. Despite the high concentrations of cis delta 9 hexadecenoate, precursor of cis delta 11 octadecenoate (vaccenate), total vaccenate levels of the five major lipid classes were lower than control values. All of these data strongly suggest that long-chain 2-ynoic acids inhibit elongation of saturated and monoene fatty acids.