Vinyl-L-NIO is a potent, selective inhibitor of nNOS. The Ki values for inhibition of nNOS, eNOS, and iNOS by vinyl-L-NIO are 100 nM, 12 and 60 μM, respectively, as determined using initial rate measurements. In the presence of NADPH and O2, vinyl-L-NIO irreversibly inactivates nNOS with a kinact of 0.078 min?1 and a Ki value of 90 nM. Vinyl-L-NIO does not inactivate iNOS. eNOS requires 20-fold higher concentrations of vinyl-L-NIO to obtain 75% the rate of inactivation seen with nNOS.