Developmental arrest in Ancylostoma caninum is associated with preparasitic, free-living third-stage (L3) larvae, as well as anthelmintic-resilient hypobiotic L3 larvae within the tissues of an infected dog. With the tissue-arrested larvae, pregnancy and, more specifically, the hormonal effects of estrogen and prolactin mediate reactivation resulting in transmammary transmission of infection to nursing puppies. Estrogen and prolactin have been shown to be critically involved in upregulation of transforming growth factor (TGF)-β2 during pregnancy, and studies on the soil nematode Caenorhabditis elegans further implicate TGF-β and insulin-like signaling pathways with larval arrest and reactivation. In this report, an in vitro assay was used to show that neither estrogen, prolactin, nor insulin had a direct effect on the feeding/reactivation response of tissue-arrested larvae; however, TGF-β isoforms 1 and 2 both had significant stimulatory effects that were comparable to the effects of dog serum. The stimulatory effects of serum could be blocked by preincubation with anti-TGF-β antibodies. Taken together, the results support the hypothesis that during pregnancy, host-derived TGF-β can signal a parasite-encoded receptor to trigger the reactivation of tissue-arrested larvae. TGF-β had no effect on preparasitic larvae, suggesting that different signals may be involved in reactivation of the 2 different arrested forms of A. caninum L3 larvae.