The Olfactomedin family is a relatively new class of extracellular proteins. Two family members have been shown to play roles in the early development of ectodermal tissues: Noelin enhances neural crest generation in chick and Tiarin promotes dorsal neural specification in Xenopus. In this study, we introduce a novel member of the Olfactomedin family, ONT1. In the early chick embryo, ONT1 expression first appears at Hensen's node and subsequently in the axial and paraxial mesoderm. When the neural tube closes, strong expression of ONT1 is transiently found in the roof plate region from the rostral midbrain to the hindbrain. Overexpression of ONT1 in these regions prolongs the generation of neural crest cells in a manner similar to that of Noelin. Interestingly, ONT1 and Noelin have opposing effects on the expression of the migrating neural crest marker HNK-1 in the chick: they, respectively, cause suppression and ectopic induction of this marker. Differential activities among Olfactomedin-related factors are further examined in Xenopus. Microinjection of ONT1 mRNA into the Xenopus embryo expands the expression domain of the neural crest marker FoxD3 at the neurula stage whereas overexpression of Tiarin or Noelin suppresses FoxD3. ONT1 exhibits no dorsalizing effects on the Xenopus neural tube, which contrasts with the strong dorsalizing activity seen for Tiarin. Thus, distinct Olfactomedin-related factors evoke qualitatively different phenotypes even in the same experimental systems, suggesting that Olfactomedin family uses multiple response systems to mediate its signals in embryogenesis.