Activation of p34^cdc2 requires a complex series of protein/protein interactions and specific phosphorylation and de-phosphorylation events to occur. The cellular role of p34^cdc2 kinase lies in the regulation of eukaryotic cell entry into mitosis, in particular into the G₂/M transition. This study examines the kinetic characterization of p34^cdc2/cyclin B kinase utilizing both the phosphocellulose filter binding assay (FBA) and a modified version of the scintillation proximity assay (SPA). Several factors were identified that elicited an effect on the kinetic constants determined for the phosphorylation reaction, with emphasis being placed on the Km apparent (Kmapp) values. Factors identified included the concentration of adenosine triphosphate (ATP) used in the reaction, the addition of a biotin label to the peptide substrate as required for capture of the phospholabeled peptide by SPA, and the source from which the p34^cdc2/cyclin B kinase was isolated. The Kmapp is the kinetic constant most frequently reported in the examination of protein phosphorylation reactions, generally being derived from simple Lineweaver-Burk analysis. In a two-substrate reaction, however, the Kmapp may not be the most informative constant, as it will be influenced by the concentration of the second substrate. In this study, true Km values were determined for ATP and a bi-otinylated peptide substrate used in the p34^cdc2 kinase-mediated phosphorylation reaction. Alberty and Dalziel constants were derived from secondary Lineweaver-Burk analysis of the phosphocellulose filter binding and SPA data. The kinetic constants determined by filter binding and scintillation proximity displayed good correlation, thus confirming the utility of scintillation proximity for the purpose of enzyme kinetic studies.