The use of three mechanism‐based probes to investigate the topology and function of fatty acid hydroxylases is discussed.1) The observation of protein rather than heme alkylation in the reaction of cytochrome P4504A1 with 10‐undecynoic acid supports the argument that the enzyme circumvents the inherent preference forω‐1 hydroxylation by restricting access to the ferryl oxygen.2) The regiochemistry of the ferricyanide‐mediated iron‐to‐nitrogen shift of the cytochrome P450102 (P450BM‐3) phenyl‐iron complex indicates that the active site of this bacterial fatty acid hydroxylase is open primarily above pyrrole ring A of the prosthetic heme group,3) Inhibition of clofibrate‐mediated peroxisome proliferation in cultured rat hepatocytes by inactivation of cytochrome P4504A1 indicates thatω‐hydroxylation of fatty acids provides a signal for peroxisome proliferation.—Ortiz de Montellano, P. R.; Chan, W. K.; Tuck, S. F.; Kaikaus, R. M.; Bass, N. M.; Peterson, J. A. Mechanism‐based probes of the topology and function of fatty acid hydroxylases.FASEB J.6: 695‐699; 1992.