RNA interference (RNAi) has become the most powerful experimental tool for the study of gene function in ticks. Subolesin, initially called 4D8, was found to be protective against tick infestations when used as a vaccine and was shown to be highly conserved among ixodid tick species at the nucleotide and protein levels. RNAi caused systemic silencing of subolesin and demonstrated that this protein is involved in regulation of tick feeding, reproduction, and development. Recently, these results were extended to the one-host tick Rhipicephalus (Boophilus) microplus in which injection of dsRNA into replete females resulted in transovarial silencing of subolesin expression in eggs and larvae. Herein, we report transovarial silencing of subolesin by RNAi in the three-host ticks, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. Silencing of subolesin expression by RNAi in these tick species also affected subolesin expression in eggs and larvae. Transovarial RNAi appears to be a common mechanism in ixodid ticks and provides a simple method for the rapid characterization of tick genes involved in oviposition, embryogenesis, and larval development.