Recent results have indicated that cAMP-dependent protein kinase (PKA) acts as a negative regulator of Hedgehog signaling in target cells of the vertebrate embryo. Consequently, suppression of PKA activity is sufficient to mimic the effect of receiving a Hedgehog signal. We have explored whether PKA-inhibiting Gi-proteins (GiPs) may also be involved in the regulation of Hedgehog signaling. Zebrafish embryos were injected with RNA encoding pertussis toxin (Ptx), a specific inhibitor of GiPs. These embryos developed phenotypic traits opposite to embryos expressing a dominant negative form of the PKA regulatory subunit (dnPKA), including a fusion of the eyes, a lack of ventral specification in the forebrain, and an expansion of the sclerotome at the expense of adaxial fates in the posterior somites. These effects can be partially rescued by coexpression of dnPKA, but not by coexpression of Indian Hedgehog, suggesting that GiPs act upstream of PKA and downstream of Hedgehogs. Other Hedgehog- and PKA-dependent processes, sclerotomal specification and adaxial specification in the first five somites, are not negatively affected by Ptx. Thus, GiPs may be involved in Hedgehog signaling in some, but not all target cells.