Mammalian pyruvate dehydrogenase (alpha 2 beta 2) (E1) is regulated by phosphorylation-dephosphorylation, catalyzed by the E1-kinase and the phospho-E1-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E1 alpha, several human E1 mutants were made with single, double, and triple mutations by changing Ser to Ala. Mutation at site 1 but not at sites 2 and/or 3 decreased E1 specific activity and also increased Km values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E1 mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E1 kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E1. However, the rates of phosphorylation and inactivation were site-specific. Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E1-phosphatase resulting in complete reactivation of the E1. The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.