l-Ascorbate, commonly known as vitamin C, serves as an antioxidant and cofactor essential for many biological processes. Distinct ascorbate biosynthetic pathways have been established for animals and plants, but little is known about the presence or synthesis of this molecule in invertebrate species. We have investigated ascorbate metabolism in the nematode Caenorhabditis elegans, where this molecule would be expected to play roles in oxidative stress resistance and as cofactor in collagen and neurotransmitter synthesis. Using high-performance liquid chromatography and gas-chromatography mass spectrometry, we determined that ascorbate is present at low amounts in the egg stage, L1 larvae, and mixed animal populations, with the egg stage containing the highest concentrations. Incubating C. elegans with precursor molecules necessary for ascorbate synthesis in plants and animals did not significantly alter ascorbate levels. Furthermore, bioinformatic analyses did not support the presence in C. elegans of either the plant or the animal biosynthetic pathway. However, we observed the complete (13)C-labeling of ascorbate when C. elegans was grown with (13)C-labeled Escherichia coli as a food source. These results support the hypothesis that ascorbate biosynthesis in invertebrates may proceed by a novel pathway and lay the foundation for a broader understanding of its biological role.