The effects of 2-hexadecynoic acid on the growth and lipid metabolism of cultured 7288 (HTC) cells have been evaluated. Growth was inhibited by the acetylenic acid: the LD50 was 35-85 microM as determined by two methods at low and high cell densities. Reduced growth did not result from damaged plasma membranes as determined by alpha-amino isobutyrate leakage. DNA synthesis was unaffected by the acetylenic acid and the effect on RNA and protein synthesis appeared to be secondary to the effects on lipid metabolism. The 2-hexadecynoic acid inhibited lipid metabolism of the HTC cells at least at two levels. Data from both mass studies and radioactive acetate distributions in cellular and media lipids indicated that fatty acid elongation and acylation, especially triglyceride synthesis, were inhibited.