Summary. Antibacterial activity, tested against Bacillus subtilis, was present in all 34 samples of human amniotic fluid collected between 37 and 41 weeks gestation. High‐molecular–weight (β‐lysin) antibacterial activity was reduced after treatment with ethylene diamine tetra–acetic acid (EDTA), but not after treatment with 1, 10‐phenanthroline or diphenylthiocarbazone. The activity was restored following incubation with magnesium, calcium, or zinc. Low‐molecular‐weight antibacterial activity (<1000 daltons) in acid–alcohol extracts was reduced after treatment with dithizone, but not after treatment with EDTA or 1, 10 phenanthroline. Activity was restored after treatment with manganese, cobalt or zinc. High‐molecular‐weight (β‐lysin) activity was therefore biochemically similar to serum p‐lysin but biochemically different from the low‐molecular‐weight antibacterial component of amniotic fluid.