However, contraction of the microfilament network is not required for membrane invagination. src64 mutant defects in both microfilament ring contraction and ring canal expansion are easily quantified, providing sensitive and effective means of assaying the biological function of src64. To understand the role of src64 in regulating microfilament ring contraction during cellularization, we identified point mutations in the src64 coding region. Of particular interest were mutations in each of the three highly conserved amino acids that constitute the HRD motif of the kinase domain catalytic loop. We analyzed the phenotypes caused by the mutation in the catalytic aspartate and the src64 null allele and found that Src64 kinase activity is required for microfilament ring contraction. We also found that mutations in the histidine and arginine residues produced weaker cytoskeletal defects and lower reductions of kinase activity than expected. We discuss the implications of these results on the roles of the HRD amino acids in kinase domain activity and activation. The catalytic loop aspartate at position 404 in Src64 has a critical role in catalysis. It has been proposed to act as a catalytic base, deprotonating the tyrosine hydroxyl to catalyze a DAPT nucleophilic attack on the c-phosphate group of ATP as part of the phosphoryl transfer reaction. However, many studies suggest that the neutral hydroxyl group acts as the nucleophile. Aspartate hydrogen bonds, directly or indirectly, to the hydroxyl group to position it for effective nucleophilic attack and acts as a proton acceptor late in the reaction. In addition, the HRD aspartate may help stabilize the inactive state through an interaction with the unphosphorylated tyrosine in the activation loop. Yeast carrying an aspartate to alanine substitution in cAMPdependent protein kinase were nearly inviable and had only 0.4% of the kinase activity of wild type. Similarly, phosphorylase kinase protein with this mutation showed little activity. Substitution with asparagine, the neutral amide derivative of aspartate, eliminates charge without altering hydrogen-bonding interactions that do not involve the carboxyl group. This mutation strongly reduced Phk kinase activity, but not as strongly as alanine. It caused a relatively small reduction in ATPase activity, suggesting that aspartate is critical for phosphoryl transfer rather than ATP hydrolysis. The asparagine mutation in the tyrosine kinase Csk also strongly reduces, but does not eliminate, kinase activity. Mutation to glutamate alters structure and size but not charge; this mutation also greatly reduces Csk activity. Mutation of the HRD arginine to alanine in yeast PKA reduces kinase activity to 10.5% of wild-type activity, but viability is unaffected. In PhK, this mutation also reduces kinase activity. Kinase activity of the chicken c-Src mutant protein is 10% for an exogenous substrate but 50% for autophosphorylation. Mutation of the HRD arginine to cysteine has remarkably little effect on Src64. Kinase activity was indistinguishable from wild type.