A high-virulence clone of serotype III Streptoccus agalactiae causing invasive neonatal disease has recently been identified by multilocus enzyme electrophoresis and can be further distinguished by its inability to grow at 40-degrees-C in a chemically defined medium. The basis for the unusual growth inhibition at 40-degrees-C was examined in the present study and shown to be owing to a temperature-sensitive fructose-1,6-bisphosphate aldolase (fba). Crude enzyme preparations (75% saturated ammonium sulfate precipitates) of fba obtained from a high-virulence clone demonstrated a 75% reduction in aldolase activity when preincubated at 40-degrees-C for 30 min compared with 37-degrees-C. In contrast, fba from a serotype III isolate obtained from an asymptomatically colonized infant demonstrated <10% decrease in activity at 40-degrees-C. Comparison of another enzyme, lactate dehydrogenase (ldh), from both organisms indicated no loss in activity at 40-degrees-C compared with 37-degrees-C. Glyceraldehyde-3-phosphate, one of the end-products of fba activity, relieved growth inhibition at 40-degrees-C.