It has been shown that apolipoprotein E allele 4 (Apo E4) represents an important risk factor in late-onset familial and sporadic Alzheimer's disease (AD) and binding of Apo E protein to one of the major players in AD, amyloid β-peptide (βA4, residues 1-43), has been demonstrated. We have tested the hypothesis that elevated intracellular free-calcium levels, leading to cell death and triggered by βA4, are raised in the presence of Apo E protein and the extent is dependent on the Apo E isoform present in the complex. We preincubated 20 nmol/l of recombinant Apo E 3 or Apo E 4, resp., either with or without 1μmol/l βA4 peptide for at least 12 hours at 37 o C. Subsequentally, intracellular free-Ca concentrations were measured after extracellular application of preincubated solutions via slow scan ratio-imaging of cultured hippocampal cells loaded with Fura-2. Interestingly, Apo E protein alone already has a moderate impact on Ca homeostasis, with Apo E4 increasing intracellular free-Ca levels to a larger extent (77 nmol/l), as compared to Apo E3 (63 nmol/l). But these effects are small as compared to the effect of Apo E/βA4 complexes. The intracellular free-Ca levels after application of Apo E/βA4 complexes were significantly higher, with Apo E4 showing highest values (434 nmol/l increase) and Apo E3 significantly less (313 nmol/l increase), but still elevated 5fold as compared to Apo E3 alone. These findings correspond to the idea that the presence of Apo E 4 alone is not enough to trigger cell death associated with AD, whereas, once βA4-peptide is present extracellularly, Apo E protein can exert its influence through Apo E/βA4 complexes. The Apo E4 allele is known to confer an earlier onset of the disease. This corresponds nicely with our finding that complexes of Apo E4 and βA4 induce the highest change in Calcium homeostasis. As carriers of the Apo E allele 3 are still susceptible to AD, we find that Apo E3 - βA4 complexes still trigger a large increase in intracellular free-Ca, which could eventually lead to AD.