The cardiac Na(+)-Ca2+ exchanger differs from most other polytopic membrane proteins in that the amino terminus is cleaved during integration into the endoplasmic reticulum membrane. In this study, the cleaved N-terminal signal sequence of the exchanger was deleted (DelSS) or rendered uncleavable by mutation of the cleavage site (MutSS). Functional analysis of the mutants expressed in Xenopus laevis oocytes and sf9 insect cells demonstrates that DelSS exchanger catalyzes Na(+)-dependent Ca2+ transport at wild-type levels, while activity of MutSS exchanger is reduced to approximately 60% of wild-type in oocytes and 20% in sf9 cells. These results indicate that neither the presence nor the cleavage of the signal peptide is required for functional assembly of the exchanger protein in the membrane. Furthermore, these observations support the concept that internal topogenic signals play the major role in membrane insertion of the Na(+)-Ca2+ exchanger.