BMP signaling is modulated by a number of extracellular proteins, including the inhibitor Chordin, Tolloid-related enzymes (Tld), and the interacting protein Twisted Gastrulation (Tsg). Although in vitro studies have demonstrated Chordin cleavage by Tld enzymes, its significance as a regulatory mechanism in vivo has not been established in vertebrates. In addition, Tsg has been reported in different contexts to either enhance or inhibit BMP signaling through its interactions with Chordin. We have used the zebrafish gastrula to carry out structure/function studies on Chordin, by making versions of Chordin partially or wholly resistant to Tld cleavage and introducing them into chordin-deficient embryos. We examined the cleavage products generated in vivo from wild-type and altered Chordins, and tested their efficacy as BMP inhibitors in the embryo. We demonstrate that Tld cleavage is crucial in restricting Chordin function in vivo, and is carried out by redundant enzymes in the zebrafish gastrula. We also present evidence that partially cleaved Chordin is a stronger BMP inhibitor than the full-length protein, suggesting a positive role for Tld in regulating Chordin. We find that depletion of the embryo for Tsg leads to decreased BMP signaling, and to increased levels of Chordin. Finally, we show that Tsg also enhances BMP signaling in the absence of Chordin, and its depletion can partially rescue the chordin mutant phenotype, demonstrating that important components of the BMP signaling pathway remain unidentified.