The amyloid-beta protein (A beta), strongly implicated in the etiology of Alzheimer's disease (AD), is formed from the amyloid-beta precursor protein (APP) through sequential proteolysis by beta- and gamma-secretases. Cleavage by gamma-secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40- and 42-amino acid A beta C-terminal variants, A beta 40 and A beta 42. The latter form of A beta is highly fibrillogenic, is invariably elevated in autosomal-dominant forms of AD, and is the major A beta component found presymptomatically in cerebral deposits. Thus, blocking production of A beta in general and A beta 42 in particular is considered an important therapeutic goal. We have developed transition-state analogue inhibitors of gamma-secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of gamma-secretase activity in APP-transfected cells, strongly suggesting an aspartyl protease mechanism; (2) gamma-secretases that form A beta 40 and A beta 42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for gamma-secretases; (4) A beta 42 production is increased several fold over control by these gamma-secretase inhibitors after replacement with inhibitor-free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total A beta and A beta 42 production hours after replacement with compound-free media and should help identify the target(s) of these protease transition-state mimics.