Autosomal dominant mutations that increase amyloid-beta(1-42) (Abeta42) cause familial Alzheimer's disease (AD), and the most common genetic risk factor for AD is the presence of the epsilon4 allele of apolipoprotein E (apoE). Previously, we characterized stable preparations of Abeta42 oligomers and fibrils and reported that oligomers induced a 10-fold greater increase in neurotoxicity than fibrils in Neuro-2A cells. To determine the effects of apoE genotype on Abeta42 oligomer- and fibril-induced neurotoxicity in vitro, we co-cultured wild type (WT) neurons with glia from WT, apoE-knockout (apoE-KO), and human apoE2-, E3-, and E4-targeted replacement (TR) mice. Dose-dependent neurotoxicity was induced by oligomeric Abeta42 with a ranking order of apoE4-TR>KO=apoE2-TR=apoE3-TR>WT. Neurotoxicity induced by staurosporine or glutamate were not affected by apoE genotype, indicating specificity for oligomeric Abeta42-induced neurotoxicity. These in vitro data demonstrate a gain of negative function for apoE4, synergistic with oligomeric Abeta42, in mediating neurotoxicity.