The Alzheimer's disease amyloid peptide Aβ has a heterogeneous COOH terminus, as variants 40 and 42 residues long are found in neuritic plaques and are secreted constitutively by cultured cells. The proteolytic activity that liberates the Aβ COOH terminus from the β-amyloid precursor protein is called γ-secretase. It could be one protease with dual specificity or two distinct enzymes. By using enzyme-linked immunosorbent assays selective for Aβ40 or Aβ42, we have measured Aβ secretion by a HeLa cell line, and we have examined the dose responses for a panel of five structurally diverse γ- secretase inhibitors. The inhibitors lowered Aβ and p3 secretion and increased levels of the COOH-terminal 99-residue β-amyloid precursor protein derivative that is the precursor for Aβ but did not alter secretion of β- amyloid precursor protein derivatives generated by other secretases, indicating that the inhibitors blocked the γ-secretase processing step. The dose-dependent inhibition of Aβ42 was unusual, as the compounds elevated Aβ42 secretion at sub-inhibitory doses and then inhibited secretion at higher doses. A compound was identified that elevated Aβ42 secretion at a low concentration without inhibiting Aβ42 or Aβ40 at high concentrations, demonstrating that these phenomena are separable pharmacologically. Using either of two methods, IC50 values for inhibition of Aβ42 and Aβ40 were found to have the same rank-order and fall on a trend line with near-unit slope. These results favor the hypothesis that Aβ variants ending at residue 40 or 42 are generated by a single γ-secretase.