Questing female blacklegged ticks, Ixodes scapularis, Say in Massachusetts rarely bear more than one endospermatophore in their reproductive tracts. We evaluated the cause of this nonrandom distribution by examining the stability of endospermatophore retention in females and the effect of prior insemination of females on the copulatory behavior of male ticks. Endospermatophores were retained without degradation in unfed female ticks for >1 yr at 5°C and for at least 4 mo at 21°C. Males were much more likely to abort preprandial (before feeding) copulations without inseminating females bearing endospermatophores. This remating inhibition activity persisted in unfed females for at least 2 mo after insemination. Perprandial (during feedng) copulations were less restrictive, particularly when females became partially engorged. Males were more likely to remain in copula with previously inseminated females that were engorged, but generally did not transfer spermatophores to them until their fifth day of attachment to a rabbit. Little inhibitory activity was evident during the final, rapid engorgement phase of feeding. Thus, remating inhibition appears to degrade as feeding progresses. In summary, an unknown factor associated with the previous insemination of females inhibits subsequent spermatophore transfer by causing mating pairs to interrupt copulation soon after initiation. Remating inhibition is strongest in unfed ticks and becomes less apparent as females become engorged.