The exploration of the combined effects of pressure and temperature leads to new insights into the structure, dynamics, and reactions of proteins. A protein molecule possesses many conformational substates, nearly isoenergetic structures performing the same function but possibly with different rates. Pressure alters both the relative substate populations and the functional properties of the individual substates and thus can produce large changes in the static and dynamic properties of a protein ensemble. Such pressure effects have been measured for myoglobin (Mb), using visible and FTIR techniques. Above a glass temperature, Tsg, pressure redistributes substate populations and changes protein structure, the position of spectral bands, and protein reaction rates. Equilbrium substate populations measured from 230 to 330 K at pressures up to 200 MPa yield a thermodynamic comparison of different substates at pH 5.5 and 6.6. Near Tsg, pressure jump experiments reveal several relaxation processes. Well below Tsg, the properties of the proteins at temperature T and pressure P depend on history, i.e., on the order in which the sample is pressurized and cooled. The effect of pressure on the binding CO and O2 to Mb after photodissociation points to the strong influence of conformational substates on protein reactions. Pressure controls the rate of rebinding through the activation volume and through redistribution of substate populations. The conformational effect of pressure differs for CO and O2, and for sperm whale and horse myoglobin. © 1990 American Chemical Society.