We have reported that a major cause of growth inhibition of Escherichia coli by L-serine is its inhibition of homoserine dehydrogenase I (HDH I), which is involved in the biosyntheses of threonine and isoleucine [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. However, Patte et al. reported that L-serine does not inhibit HDH I [Patte, J.-C., Truffa-Bachi, P., & Cohen, G. N. (1966) Biochim. Biophys. Acta 128, 426-439]. In studies on the reason for these discrepant results, we found that the concentration of K⁺ and the pH in the assay mixture strongly influenced the inhibitory effect of L-serine. L-Serine strongly inhibited the HDH I activities in both the forward and reverse reactions between aspartate semialdehyde and homoserine at a physiological K⁺ concentration (100 to 200mM) and physiological pH (7.5) for E. coli cells. On the other hand, two well-known inhibitors of HDH I, L-threonine and L-cysteine, strongly inhibited the activity regardless of the K⁺ concentration and pH.