Cells ofStreptococcus faecalisthat survived heating for 21 min at 60 °C were killed when resuspended at an initial cell density of about 1 × 108viable units/mL and incubated at 33 °C for 24 h in a no-growth medium containing potassium phosphate buffer, pH 7.1, glucose, and casein hydrolysate. When such heated cells were resuspended at an initial cell density of about 1 × 107viable units/mL or lower, subsequent cell death was reduced at least 10 000-fold. Unheated cells incubated under similar conditions at about 1 × 108and 1 × 109viable units/mL did not die. Cell death was due to a toxic compound synthesized by the heated cells, and supernatants from incubations showing a bactericidal effect contained a component, absent in nonlethal supernatants, that reacted with 2,4-dinitrophenylhydrazine. Thin-layer chromatography, mass spectrometer analysis, and the visible spectrum of the 2,4-dinitrophenylhydrazine derivative of the unknown and authentic methylglyoxal, and the positive response shown by the free unknown compound when used as a substrate for glyoxalase I, suggested that methylglyoxal was the bactericidal compound. Solutions of authentic methylglyoxal were bactericidal at concentrations above 0.2 mMand lethal supernatants contained about 1 mMmethylglyoxal, whereas supernatants that were not lethal contained <0.02 mMmethylglyoxal.