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To determine the levels and biochemical characteristics of substance P–like immunoreactivity (SPLI) in human tears and ascertain whether substance P (SP) concentrations in tears reflect the condition of the ocular surface.Unstimulated tears were collected with a micropipette. Tear samples were partially purified using C-18 cartridges. Levels of SPLI in purified samples were measured using an enzyme immunoassay (EIA). For biochemical characterization of SPLI, tear extracts were fractionated using high-performance liquid chromatography (HPLC); each fraction was then subjected to EIA. To determine the catabolism of SP in tears, synthetic SP was incubated in medium containing pooled tears and then analyzed using HPLC.The concentration of SPLI in normal human tears was 306.0 ± 96.5 pg/mL (mean ± SD, range 148–555 pg/mL). Levels of SPLI did not vary significantly by age or gender. Concentrations of SPLI in tears from eyes with unilateral corneal hypesthesia were lower than those in tears from contralateral healthy eyes. Diclofenac sodium eye drops reduced concentrations of prostaglandin E2 and SPLI in tears. Analysis using HPLC indicated that five different substances contributed to SPLI in tears and that SP was broken down into several fragments, including SP8–11, by enzymes present in tears.Substance P is a normal component of human tears. Levels of SPLI in tears might reflect the denervated status of the ocular surface. Substance P is catabolized by degradative enzymes in tears to maintain the ocular surface by exerting the trophic effects of SP while avoiding undesirable effects.