CTP:phosphocholine cytidylyltransferase α (CCTα) contains a central region that functions as a catalytic domain, converting phosphocholine and cytidine 5′-triphosphate (CTP) to CDP-choline for the subsequent synthesis of phosphatidylcholine. We have investigated the catalytic role of lysine 122 and arginine 196 of rat CCT" using site-directed mutagenesis and a baculovirus expression system. Arginine 196 is part of the highly conserved RTEGIST motif, while lysine 122 has not previously been identified by protein sequence alignment as a candidate catalytic amino acid. Removing the side chain of lysine 122 compromises the catalytic ability of CCT", decreasing the apparent Vmax value in mutant enzymes Lys122Ala and Lys122Arg to 0.30 and 0.09% of the wild-type value, respectively. The decrease in Vmax is accompanied by dramatic 471- and 80-fold increases in the apparent Km value for phosphocholine but no greater than 3-fold increases in the apparent Hill constant (K*) value for CTP. Mutation of arginine 196 to lysine results in an enzyme that retains 24% of the wild-type Vmax value with a modest 5-fold increase in the Km value for phosphocholine. However, the Arg196Lys mutant enzyme exhibits a 23-fold increase in the K* value for CTP. These data suggest lysine 122 and arginine 196 of rat CTP:phosphocholine cytidylyltransferase are functionally important amino acids, perhaps at or near the active site involved in forming contacts with the substrates phosphocholine and CTP, respectively.