The hematogenous and the ascending routes of infection are mainly discussed in the pathogenesis of pyelonephritis (PN). Animal experiments were initiated to study the efficiency of either route and the influence of additional factors, such as virulence of the infecting organisms, application of hormones, or mechanical lesion on the kidney. The ascending route of infection was not effective without additional facilitating factors, except for the E. coli O2 strain; due to application of hormones (estrogen, progesterone, prednisolone) PN occurred with serum resistant strains in a significant number of kidneys, but not with serum-sensitive strains. Medication of hormones or microtrauma (endovesical instillation of 1 ml of sterile saline) alone did not render the hematogenous route of infection effective, whereas the combination of both hormone and microtrauma resulted in a high incidence of renal infection. Ligature of the ureter made the kidneys equally susceptible to infection by both routes, irrespective of the virulence of the E. coli strain. It is concluded that in the pathogenesis of PN both the hematogenous and the ascending routes of infection are possible. The E. coli organisms will take the one pathway or the other depending on the infection facilitating factors that prevail in the individual case.