APH-1 is one of the four essential components of presenilin (PS)-gamma-secretase complexes. There are three major isoforms of APH-1 in humans: APH-1aS, APH-1aL, and APH-1b. To gain insight into the functional role of APH-1 in gamma-secretase complexes, we analyzed the relationship between the three APH-1 forms and characterized APH-1 mutants with a disrupted transmembrane GxxxG motif. We found that overexpression of APH-1aS or APH-1b in human cells significantly reduced the levels of endogenous APH-1aL protein. However, this displacement was not observed in PS-deficient cells, suggesting that it is dependent on PS. In transiently transfected cells, the levels of APH-1aL with G122D or L123D mutations were much lower than wild-type APH-1aL. Also, cycloheximide treatment of stable transfectants revealed that the mutant proteins are much less stable than the wild type. Furthermore, coimmunoprecipitation analysis showed that wild-type but not the mutant APH-1aL is incorporated into PS1 complexes, displacing endogenous APH-1aS. These results collectively indicate that the three forms of APH-1 can replace each other in PS complexes and that the transmembrane GxxxG region is essential for the stability of the APH-1 protein as well as the assembly of PS complexes.