The phosphorylation of glyoxalase I was observed when the phosphatase-treated enzyme was incubated in the presence of [gamma-32P]ATP and a cell extract prepared from alpha-type yeast cells which had been treated with the culture supernatant of a-type yeast cells. The phosphorylated protein was identified as glyoxalase I by using anti-glyoxalase I rabbit immunoglobulin G.