The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-penteonic acid on the in incorporation of label from [1-14C]acetate, [2-14C]glycerol, [1-14C]ribose, [U-14C]fructose, or [1-14C]glucose into CO2 but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest approximately 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-14C]acetyl CoA into glycogen, lipids, CO2, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit beta-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments.