p38alpha and p38beta MAPKs (mitogen-activated protein kinases) share about 80% of their protein sequence identity, but have quite different biological functions. One such difference is in regulating the subcellular localization of their downstream kinases, such as PRAK (p38-regulated/activated protein kinase or MK5). The p38alpha-PRAK complex is found in the nucleus, whereas the p38beta-PRAK complex is exclusively localized to the cytosol. By generating a series of chimeric and point mutants of p38alpha and p38beta, we found two amino acid residues (Asp(145) and Leu(156) in p38alpha, Gly(145) and Val(156) in p38beta) that determine the distinct subcellular locations of p38alpha-PRAK and p38beta-PRAK. The subcellular localization of MK2 (MAPK-activated protein kinase 2), another downstream kinase of p38, was regulated in the same manner as that of PRAK. We found that nuclear import, but not export, determines the subcellular localization of p38alpha-PRAK and p38beta-PRAK. The published structure of the p38alpha-MK2 complex suggests Leu(156) of p38alpha is involved in the interaction with the nuclear localization signal in PRAK. The difference at this residue between p38alpha and p38beta may affect the nuclear localization signal in PRAK differently, and thereby influence the import of the complexes. Asp(145) in p38alpha (or Gly(145) in p38beta) is located on a different surface patch, and further random mutagenesis revealed that mutation of Asp(145), Thr(123), and Gln(325), the residues that can directly interact with importin alpha as predicted by modeling, but not mutation of the other 7 amino acid residues that cannot reach importin alpha, re-locate p38alpha-PRAK to the cytosol, suggesting that interaction with import machinery is involved in determining the subcellular localization of the p38alpha-PRAK and p38beta-PRAK complexes. Last, we show that nuclear localization of PRAK is required for its role in inhibiting the proliferation of NIH3T3 cells. In conclusion, multiple determinants control the distinct subcellular localization of p38alpha-PRAK and p38beta-PRAK complexes, and the location of PRAK plays a role in its function.