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.