Therapy of chronic E. coli pyelonephritis in rats was equally effective with cyclacillin and oral ampicillin, whereas intramuscular ampicillin had a significantly higher therapeutic activity than oral cyclacillin. Serum concentrations in rats and mice were consistently higher with cyclacillin than with ampicillin and showed great variations depending on the animal species. It was concluded that there is a good correlation between in vivo and in vitro activity of both antibiotics, provided that the serum levels are taken into consideration. On this basis it may be predicted that in man cyclacillin will exhibit lower therapeutic activity in gram-negative infections than ampicillin.