Ecdysteroids are the only hormones unequivocally identified thus far in ticks. We found a positive correlation between ecdysteroid concentration and vitellogenin synthesis in female Ixodes scapularis. Vitellogenin (Vg) synthetic activity was measured by an in vitro assay for Vg, involving incubations of the fat body with 35S-methionine and immunoprecipitation collected on a solid-phase matrix, protein A. Vitellogenin synthetic activity in the fat body was undetectable in unfed females but was detected after tick attachment to the host. Vitellogenin production in the fat body remained low from attachment until 2 days prior to detachment from the host. Vitellogenin synthesis in the fat body peaked 2 days after detachment and declined to a level 2-3 times above background from 6 days after dropping from the host through oviposition. A peak of ecdysteroids in females 6 days after attachment preceded an increasing rate of Vg synthesis, suggesting a positive correlation between these parameters. Ecdysone and 20-hydroxyecdysone, the 2 major ecdysteroids present during vitellogenesis, could not be detected in females prior to feeding or 2 days after attachment to the host; however, concentrations began to increase 4 days after attachment, peaked during rapid engorgement, and subsequently declined.