Escherichia coli 536 (O6:K15:H31), which was isolated from a case of urinary tract infection, determines high nephropathogenicity in a rat pyelonephritis system as measured by renal bacterial counts 7 days after infection. The loss of S fimbrial adhesin formation (Sfa-) (mannose-resistant hemagglutination [Mrh-] and fimbria production [Fim-]), serum resistance (Sre-), and hemolysin production (Hly-) in the mutant 536-21 led to a dramatic reduction of bacterial counts from almost 10(5) to only 40 cells per g of kidney. The reintroduction of the cloned S fimbrial adhesin determinant (sfa) increases the virulence of the avirulent mutant strain by a factor of 20; almost the same effect was observed after restoration of serum resistance by integration of an sfa+ recombinant cosmid into the chromosome. Additional reintroduction of the Hly+ phenotype by transformation of two hly determinants increased the virulence of the strains. Hemolysin production determined increased renal elimination of leukocytes and erythrocytes. Thus all three determinants investigated, S fimbriae, serum resistance, and hemolysin, contribute to the multifactorial phenomenon of E. coli nephropathogenicity.