C₁ metabolism in plants is known to be involved in photorespiration, nitrogen and amino acid metabolism, as well as methylation and biosynthesis of metabolites and biopolymers. Although the flux of carbon through the C₁ pathway is thought to be large, its intermediates are difficult to measure and relatively little is known about this potentially ubiquitous pathway. In this study, we evaluated the C₁ pathway and its integration with the central metabolism using aqueous solutions of 13C-labeled C₁ and C₂ intermediates delivered to branches of the tropical species Inga edulis via the transpiration stream. Delivery of [13C]methanol and [13C]formaldehyde rapidly stimulated leaf emissions of [13C]methanol, [13C]formaldehyde, [13C]formic acid, and 13CO₂, confirming the existence of the C1 pathway and rapid interconversion between methanol and formaldehyde. However, while [13C]formate solutions stimulated emissions of 13CO₂, emissions of [13C]methanol or [13C]formaldehyde were not detected, suggesting that once oxidation to formate occurs it is rapidly oxidized to CO₂ within chloroplasts. 13C-labeling of isoprene, a known photosynthetic product, was linearly related to 13CO₂ across C₁ and C₂ ([13C₂]acetate and [2-13C]glycine) substrates, consistent with reassimilation of C₁, respiratory, and photorespiratory CO₂. Moreover, [13C]methanol and [13C]formaldehyde induced a quantitative labeling of both carbon atoms of acetic acid emissions, possibly through the rapid turnover of the chloroplastic acetyl-CoA pool via glycolate oxidation. The results support a role of the C₁ pathway to provide an alternative carbon source for glycine methylation in photorespiration, enhance CO₂ concentrations within chloroplasts, and produce key C₂ intermediates (e.g., acetyl-CoA) central to anabolic and catabolic metabolism.