A method was developed for urinary nitrate analysis utilizing an enzyme of Escherichia coli for the reduction of nitrate to nitrite. The resulting nitrite was assayed by a standard diazotization procedure. Under the experimental conditions stoichiometric conversion of nitrate to nitrite was achieved. Crude enzyme present in bacterial suspensions was used without any initial purification and no prior treatment of the urine samples was necessary. The bacteria were cultured under conditions producing high nitrate reductase activity and used formate as an exogenous electron donor without demonstrating any nitrite reductase activity. The procedure was subsequently automated to produce rapid, simultaneous determination of urinary nitrate and nitrite at the rate of 45 analyses/hr.