The intrinsic ability of human amniotic fluid to prevent growth of Escherichia coli varies with the test strain employed, but is enhanced by increasing the temperature of incubation, as demonstrated by viable counts of test organisms in amniotic fluid compared with growth media at various temperatures. The antibacterial activity of amniotic fluid is reversed at all temperatures by the addition of potassium phosphate, but residual inhibition is seen at temperatures above 37C. When purified inhibitor preparations are tested, complete reversal by phosphate at the highest temperatures is seen. Thus, the augmented inhibition in whole amniotic fluid depends on the combined effects of phosphate-sensitive and -insensitive inhibitors. Test organisms in the presence of partially purified inhibitor were not adversely affected at 4C, indicating that bacteria must be metabolically active to be inhibited. It is concluded that temperatures above 37C enhance the antibacterial effect. This may enhance protection during pyrexia.