This study provides strong evidence against cytochrome b5 participation in the first reduction step-beta-ketoreduction-of rat liver microsomal fatty acid chain elongation. Several lines of evidence led to this conclusion: (a) beta-ketoreductase was not inducible by diet conditions since its activity was the same in microsomes from fasted rats and in rats fed a fat-free diet. Consequently, its activity was appreciable in microsomes from fasted rats. Nevertheless, cytochrome b5 reoxidation rate was not stimulated by adding beta-ketopalmitoyl-CoA to the latter microsomes. This suggests that it is not the activated beta-ketoreductase which stimulates the cytochrome b5 reoxidation rate, but another electron acceptor. (b) The delta 9-desaturase, present in microsomes from rats fed a fat-free diet, was totally inhibited by 4 mM KCN; beta-ketopalmitoyl-CoA or malonyl-CoA stimulated the reoxidation rate of cytochrome b5 but this increase was also inhibited by 4 mM KCN. This suggests that delta 9-desaturase is involved in the stimulation and shows that any inhibitor of delta 9-desaturase, including cytochrome b5 antibodies, may induce elongation inhibition. (c) NADH-dependent beta-ketoreductase activity was partially purified from Triton X-100 solubilised microsomes, in a fraction essentially free of cytochrome b5. Furthermore, when the fraction containing cytochrome b5 and NADH-cytochrome-b5 reductase was added to the fraction containing beta-ketoreductase activity, no increase in beta-ketoreductase activity was observed. Stearoyl-CoA desaturase activity which is also present in microsomes from rats fed a fat-free diet led to the results which have been misinterpreted in the conclusions of previous studies.