Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the γ-secretase complex and cleave many type I transmembrane proteins including β-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause ‘abnormal’ gain or (partial) loss of function of γ-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on γ-secretase complex formation. A loss in APP and Notch substrate processing at ɛ and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the γ site was affected in variable ways. PS1-Δ9 and PS1-L166P mutations caused a reduction in β-amyloid peptide (Aβ)40 production whereas PS1-G384A mutant significantly increased Aβ42. Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Aβ than PS1. Finally, subtle differences in γ-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect γ-secretase structure or function in multiple ways.