The native state ensemble of a protein is comprised of distinct conformational substates in equilibrium, exchanging on the μs - ms time scale. Functionally important substates can be sparsely populated (“excited states”) and invisible to conventional spectroscopic methods, but recent studies have shown that high hydrostatic pressure can populate excited states and slow exchange kinetics to the easily measurable millisecond and longer time window. Thus, hydrostatic pressure can populate excited states for spectroscopic characterization, and pressure-jump relaxation experiments can determine the exchange rates between excited and ground states. Site Directed Spin Labeling (SDSL) - Electron Paramagnetic Resonance (EPR) spectroscopy of proteins containing a paramagnetic nitroxide side chain (a “spin label”) has been shown to provide information on both structure and dynamics in proteins of any degree of complexity. This makes it an ideally suited spectroscopic technique for these studies. Here we present a pressure-jump EPR system that achieves sub-millisecond pressure jumps or drops of arbitrary magnitude. We use a pressure intensifier capable of generating hydrostatic pressures up to 4 kbar (60,000 psi) to pressurize two reservoirs, and a novel air operated valve to rapidly equilibrate the reservoirs to an intermediate final pressure. Pressure cells and resonators have been developed for both X and Q band operation; the Q band system employs a modified Varian E-110 Q-band bridge. The pressure-jump system and EPR spectrometer are controlled via a user interface constructed with Labview. Together, these components comprise a complete pressure-jump EPR system for quantifying millisecond or slower conformational exchange rates in proteins. Preliminary results on conformational exchange rates in apo-myoglobin and T4 Lysozyme cavity mutants will be presented.