We used amino acid (AA) compound-specific isotope analysis (d15NAA and d13CAA values) of midwater zooplankton and suspended particles to examine their dynamics in the mesopelagic zone. Suspended particle d15NAA values increased by up to 14% with depth, whereas particle trophic status (measured as trophic position, TP) remained constant at 1.6 6 0.07. Applying a Rayleigh distillation model to these results gave an observed kinetic isotope fractionation of 5.7 6 0.4%, similar to that previously measured for protein hydrolysis. AA-based degradation index values also decreased with depth on the particles, whereas a measure of heterotrophic resynthesis (SV) remained constant at 1.2 6 0.3. The main mechanism driving 15N enrichment of suspended particles appears to be isotope fractionation associated with heterotrophic degradation, rather than a change in trophic status or N source with depth. In zooplankton the ‘‘source’’ AA phenylalanine (Phe) became 15N enriched by up to 3.5% with depth, whereas zooplankton TP increased by up to 0.65 between the surface ocean and midwaters. Both changes in the d15N values of food resources at the base of the zooplankton food web and changes in zooplankton TP drive observed zooplankton 15N enrichment with depth. Midwater zooplankton d15NPhe values were lower by 5–8% compared with suspended particles, indicating this organic matter pool is not a significant zooplankton food resource at depth. Instead, 62–88% of the N sustaining midwater zooplankton is surface derived, obtained through consumption of sinking particles, carnivory of vertical migrants, or direct feeding in surface waters at night.