Proven isogenic capsule-negative derivatives (CP9.29, CP9.108, CP9.137, CP9.171, CP9.443, and CP9.C56), generated from an O4/K54/H5 blood isolate (CP9) of Escherichia coli by IS50L::phoA (TnphoA)-mediated transposon mutagenesis, were used to assess the function of a non-K1 capsule in three animal models. Intraperitoneal injection of CP9 (K54+) into mice resulted in an LD50 at 24 h of 5.5 x 106 cfu compared with LD50s of 2.6 x 107 cfu and 3.8 x 107 cfu for CP9.108 (K54-) and CP9.C56 (K54-) (P < .001). CP9 was cleared less rapidly from the bloodstream, after intravascular injection, than was CP9.108 (P < .01). In the rat granuloma pouch model, CP9 could proliferate from starting inocula as low as 1.0 x 103 cfu/mL. In contrast, capsule-deficient derivatives underwent transient log kills with starting inocula as high as 1.0 x 106 cfu/mL. Because proven isogenic strains were evaluated, a clear contribution of the K54 capsular polysaccharide to virulence in vivo is demonstrated.