The effect of water restriction and ammonium chloride acidosis on the course of Escherichia coli pyelonephritis was determined in the nonobstructed kidney of the rat. To alter the chemical composition of the renal medulla, water intake was reduced in rats to one-half the normal daily intake. Water restriction increased the incidence of coliform pyelonephritis. Systemic acidosis, produced by giving a 300 mM solution of ammonium chloride, increased urinary osmolality to values comparable to water restriction and also predisposed to pyelonephritis. However, when rats were fed the same solution of ammonium chloride but were allowed access to tap water ad lib., urinary osmolality values were comparable to those observed in normal animals, and susceptibility to pyelonephritis was reduced or eliminated despite a degree of systemic acidosis similar to that observed in rats fed ammonium chloride solution without access to tap water. These results suggest that water diuresis may overcome the inactivation of complement produced by ammonium chloride acidosis and that renal medullary hypertonicity, produced by either water restriction or ammonium chloride acidosis, is a major determinant of this tissue's unique susceptibility to infection.