Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.