The substrate-bound structures of two cytochrome P450s, P450cam and P450eryF, are known. While these structures reveal important features that control substrate specificity, the problem of how conformational changes allow for substrate entry and product release remains unsolved. The structure of the haem domain of the bacterial fatty acid hydroxylase, P450BM-3, previously was solved in the substrate-free form. Unlike the substrate-bound P450cam and P450eryF structures, the substrate access channel is open in substrate-free P450BM-3. Here we present the X-ray structure of P450BM-3 at 2.7 A bound with a fatty acid substrate, palmitoleic acid. A comparison of the substrate-bound and -free forms reveals major conformational differences and provides the first detailed picture of substrate-induced conformational changes in a P450.