The γ-secretase enzyme complex is responsible for the cleavage of amyloid precursor protein (APP) to form the 40 or 42 amino acid amyloid beta protein (Aβ). Inhibition of γ-secretase has been suggested as a potential therapy in Alzheimer’s disease however we have previously shown that inhibition of this enzyme complex can be toxic, resulting in a 60% loss of viability in primary neuronal cultures and neurone-like cell lines (Plant et al. 2003). In this study we have used three inhibitors of γ-secretase which differentially decrease production of the two size forms of amyloid beta protein and have investigated the resulting effects on cell viability. Dissociated cultures of cortical neurones were prepared from 1 day old rats and maintained as reported previously (Plant et al. 2005). SH-SY5Y cells were cultured as reported previously (Plant et al. 2005). Data are presented as mean ±S.E.M and statistical differences were assessed using Student’s t test. Human neuroblastoma SH-SY5Y cells and primary cortical neurones were incubated with 10μM γ-secretase inhibitor for 24 hours. Preferential inhibition of release of Aβ_1-40, using γ-secretase inhibitor I, resulted in a 56.5±5.3% (n=7, P<0.001) reduction in viability in SH-SY5Y cells and a 29±15% (n=3) reduction in cell viability in primary cortical neurones as measured using a Live/Dead cytotoxicity assay kit. Selective inhibition of production of the 42 amino acid isoform, using γ-secretase inhibitor VI, had no significant effect on viability in either cell type. Inhibition of release of both peptides concurrently by γ-secretase inhibitor IV resulted in an 8.8±3.2% decrease in viability (n=13, P<0.05) in SH-SY5Y cells and a 15.0±4.9% decrease in viability of cortical neurones (n=3). Co-incubation of cells with 1nM Aβ_1-40 partially reversed the effects of inhibiting production of Aβ_1-40 (12.8±14.0% increase in viability, n=6) and completely reversed the toxicity associated with preventing production of Aβ_1-40 and Aβ_1-42 concurrently (viability returned to 99.7±0.5%, P<0.001, n=6). Enhanced differentiation of SH SY5Y cells by exposure to 10μM retinoic acid for 7 days resulted in exaggerated responses to the secretase inhibitors (n=2). These findings demonstrate that inhibition of production of amyloid beta _1-40 but not _1-42 is detrimental to cells suggesting a pivotal role of amyloid beta _1-40 in normal neuronal physiology.