A systematic mutagenesis strategy was used to identify the functional regions and residues of a protein kinase. Clusters of the charged amino acids in the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase, were systematically mutated to alanine, producing a set of mutations that encompassed the entire molecule. Residues indispensable for enzyme activity were identified by testing the ability of the mutants to function in vivo. Active mutants were assayed in vitro, and mutants with reduced specific activity were subsequently analyzed by steady-state kinetics to determine the effects of the mutation on kcat and on Km for MgATP and for a peptide substrate. Specific residues and regions of the enzyme were identified that are likely to be important in catalysis and in binding of MgATP, functions that are common to all protein kinases. Additional regions were identified that are likely to be important in binding a peptide substrate, the recognition of which is likely to be specific to the serine/threonine protein kinases that have a requirement for basic residues around the target hydroxyamino acid. The properties of mutants defective in substrate recognition were consistent with an ordered sequential reaction mechanism. This represents the first comprehensive analysis of a protein kinase by a rational mutagenesis strategy.