Cytochrome P450 BM3, of bacterial origin, is one of only five isozymes of the ubiquitous family of over 400 metabolizing heme proteins with a known crystal structure and only one of two with both substrate-free and substrate-bound forms determined. P450 BM3 is of particular interest since it has a similar function and similar substrates as mammalian P450s particularly of the 4A subfamily. Thus, the extent to which the substrate-free form of P450 BM3 undergoes a conformational change upon binding of a typical fatty acid substrate, palmitoleic acid, has been the subject of recent active experimental effort. Surprisingly, direct examination of the substrate-free (pdb2hpd.ent and pdb2bmh.ent) and substrate-bound (pdb1fag.ent) forms do not provide a clear answer to this question. The main reason for this ambiguity is that the two substrate-free monomers reported in the crystal structures themselves have significantly different conformations from each other, one with a more open substrate-access channel than the other. Since there is no way to tell to which substrate-free form the substrate binds, the effect of substrate binding cannot be deduced directly from comparisons of the experimental substrate-bound and substrate-free forms. The computational studies reported here have been designed to more robustly establish the effect of substrate binding on this isozyme. Specifically, molecular dynamics simulations were performed for each of the two substrate-free forms found in the asymmetric unit of the X-ray structure and for the two corresponding substrate-bound forms, constructed by docking palmitloeic acid into each of them. Comparisons of the results showed that palmitoleic acid binding had little effect on the conformation of the more closed substrate-free form of P450 BM3. By contrast, in the more open substrate-free form, this same substrate induced a closing of the entrance to the substrate-binding channel. The MD averaged structure of these two complexes obtained from docking of pamitoleic acid into the two asymmetric units of the substrate-free form were also compared to that obtained starting with the X-ray structure of the substrate-bound form. These results taken together led to the conclusion that, if indeed the substrate induces conformational changes in P450 BM3, the mouth of the substrate-access channel first closes down in response to the presence of the substrate, followed by rotation of the F-G domain to further optimize the P450 BM3-substrate interaction that would occur at a later stage.