<jats:p> We have partially purified homoserine kinase from a genetically derepressed strain of <jats:italic>Escherichia coli</jats:italic> K-12. The optimum pH of the enzyme-substrate reaction was 7.8 and the <jats:italic> K <jats:sub>m</jats:sub> </jats:italic> values for <jats:sc>l</jats:sc> -homoserine and adenosine 5′-triphosphate were both 3 × 10 <jats:sup>−4</jats:sup> M. K <jats:sup>+</jats:sup> (or NH <jats:sub>4</jats:sub> <jats:sup>+</jats:sup> ) as well as Mg <jats:sup>2+</jats:sup> were required for its activity. The sedimentation coefficient determined by ultracentrifugation in a sucrose density gradient was 5.0 ± 0.25 <jats:italic>S</jats:italic> . <jats:sc>l</jats:sc> -Homoserine was an excellent protector against heat inactivation of homoserine kinase. <jats:sc>l</jats:sc> -Threonine was a competitive inhibitor of homoserine kinase, suggesting that end-product inhibition of this enzyme plays a role in vivo in the overall regulation of threonine biosynthesis. The specific activity of aspartokinase I-homoserine dehydrogenase I and of homoserine kinase showed a strong positive correlation in extracts from strains under widely varying conditions of genetic or physiological derepression; it was concluded that these two enzymes are coordinately regulated in <jats:italic>E. coli</jats:italic> K-12.