The effect on sperm motility of nonoxynol-9 chlorhexidine diacetate was compared in semen and cervical mucus. Both compounds had similar spermicidal potency in semen, abolishing sperm motility within 3 minutes at 0.5 mg/ml. When these compounds were allowed to diffuse into mucus, the subsequent survival of sperm in the mucus was different. Restricted penetration and loss of motility occurred rapidly after treatment with 0.1 mg/ml chlorhexidine, whereas sperm survived normally in mucus after prolonged contact with 200 mg/ml chlorhexidine. When the compounds were mixed directly with the mucus before sperm penetration was attempted, chlorhexidine still immobilized sperm, but concentrations of nonoxynol-9 that would be spermicidal in semen had no effect in mucus.