A series of pepstatin analogues having minimum structural requirements for tight-binding inhibition has been synthesized and tested on porcine pepsin. Subtle changes in the geometry and size of side chains at the valine-1 position of pepstatin were found to dramatically affect inhibitor potency as well a the type of kinetic behavior observed. The inhibitors reported here can be grouped into two categories: the more potent inhibitors are slow-binding inhibitors, i.e., exhibit slow, time-dependent inhibition: the weaker inhibitors, with Ki values greater than 10(-8) M, are not time-dependent inhibitors. A minimum kinetic mechanism is proposed to account for the observed kinetic behavior.