pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. it is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4-to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2-10.5 nmol/cm 2), propionic acid (0.1-8.5 nmol/cm 2), glycine (0.7-3.5 nmol/cm 2), serine (0.08-2.3 nmol/cm 2), galactose (galactose + mannose; 0.035-0.078 nmol/cm 2), lactose (0.002-0.086 nmol/cm 2), glucose (0.018-0.953 nmol/cm 2), palmitic acid (0.26-2.03 nmol/cm 2), and stearic acid (0.34-1.81 nmol/cm 2). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples. Despite a general worldwide decline, tooth decay still remains a severe problem for both health and the economy. In 2010, the cost for medical treatment of dental diseases was USD 298 billion worldwide, corresponding to 4.6% of the total global health-care expenditure 1. Although considered preventable, caries is one of the most prevalent non-communicable diseases in Europe, with up to 100% of the adults having an experience with it 2. The disease can arise already in the first years of life and challenges the physiological and psychological health of both children and their parents 3,4. Furthermore, 621 million children are affected worldwide 5. Up to 26% of 3-year-olds in Germany already show the initial stage of early childhood caries (ECC) or nursing bottle syndrome 6. ECC is defined as the presence of one or more decayed, missing, or filled tooth surfaces in any primary tooth in a child ages ≤6 years and can be characterised, for example, by the dmf/s = decayed, missing, filled surface (dmf/s) or decayed, missing, filled teeth (dmf/t) index 7. The development of caries in general and ECC in particular is a multifactorial process 8 , with contributions found in irresponsible feeding practice with unrestricted access to sweetened beverages especially at night, presence of cariogenic microorganisms, genetic factors and in particular inadequate oral hygiene habits 3. The physical damage of the teeth, e.g. lesions and cavities, is considered the final and visible stage within the progression of this complex illness 9. Aside from local pain, untreated caries can successively evolve from lesions further ...