A genetic dimorphism incorporates either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence (MTS) of manganese superoxide dismutase (MnSOD). The Ala-MTS confers a 40% higher MnSOD activity than the Val-MTS after import into isolated mitochondria in vitro. The present study aimed to characterize functional consequences in whole cells. HuH7 human hepatoma cells were transfected with vectors encoding for the human Ala- or Val-MnSOD variants fused to a Myc-His-tag. The Ala-variant resulted in four-fold higher levels of the mature exogenous protein and MnSOD activity than the Val-variant. Studies with a proteasome inhibitor indicated that precursor proteins are either imported into the mitochondria or degraded by the proteasome. Despite identical levels 8 h after transfection, mRNA levels at 36 h were two-fold higher for the Ala-encoding mRNA than the Val-mRNA. Decreasing the mitochondrial membrane potential decreased both MnSOD mitochondrial import and its mRNA levels. Much larger differences in the activity of the human Val- and Ala-MnSOD variants are observed in whole cells rather than after import experiments performed in vitro. First, the slowly imported Val-MnSOD is degraded by the proteasome in cells. Second, the slower mitochondrial import of the Val-variant may be associated with decreased mRNA stability, possibly due to impaired cotranslational import.