Studies are presented on the isolation, localization, and characterization of hippuricase activity of group B streptococci. Washed, intact cells, live or heat killed at 56 C, exhibited hydrolysis of hippuric acid, but cell-free filtrates of the organism did not. Excellent hippuricase activity was recoverable from supernatant fluids of mechanically disrupted cells, and evidence suggests that it exists largely intracellularly. Characteristics of the hippuricase preparation are consistent with the view that the biologically active principle is an enzyme. A quantitative microtiter technique has been developed which is useful in titrating enzymatic activity and antibody neutralization. Sera from rabbits immunized with filtered preparations neutralized hippuricase activity.