Conserved histidine residues have been implicated in the geometry and catalytic mechanism of the E(1)alpha proteins of the PDH complex. We constructed and expressed a series of PDH-E(1)alpha histidine mutants (H63, H84, H92, and H263) in a cell line with zero PDH complex activity due to a null E(1)alpha allele, Based on immunoblot and enzyme activity analyses, all introduced histidine mutations, with the exception of H92, affected the specific activity of the PDH complex. We showed that H63 and H263 are essential for the activity since mutations introduced at those sites produced a PDH complex with near-zero activity. Mutations introduced at H84 only partially reduced activity, implying that H84 may play a less critical role in the PDH complex. Mutations introduced at H92 caused the absence of immunoreactive material for both the E(1)alpha and E(1)beta subunits and may have impaired import or assembly of precursor peptides into the mature PDH complex.