The main goal of our study was to analyze and compare the profiles of secreted proteins from adult human articular chondrocytes in monolayers, and cartilage explants in culture, using a de novo protein labeling approach. Stable isotope labeling of proteins in culture was used to differentiate between chondrocyte-derived proteins and other preexisting matrix-derived components, or proteins coming from serum or synovial fluids. Proteins in culture supernatants were resolved by one-dimensional SDS-PAGE electrophoresis, and analyzed in tandem with LC/MS-MS (liquid chromatography/double mass spectrometry). Results from stable isotope labeling with amino acids in cell culture (SILAC) were validated by specific immunoblotting of four relevant proteins identified in the secretion media. After 8-10 days of culture, over 90% of proteins secreted during monolayer growth contained (13)C(6)-Arg and (13)C(6)-Lys. Nonlabeled proteins corresponded mostly to plasma-associated proteins, indicating background contamination of medium with serum remnants. The majority of the secreted proteins in 2D cultures were extracellular matrix components and matrix regulators, along with some inflammatory agents and metabolic enzymes. In explants, only 25%-30% of proteins were labeled with heavy amino acids, corresponding to matrix regulators and carrier molecules. Nonlabeled proteins corresponded primarily to structural matrix components. In qualitative terms, all labeled proteins coming from cartilage explants were also found in chondrocytes supernatants. In summary, our results show differences in the labeling pattern of proteins found in supernatants from explants and monolayers. Most proteins found in the media of explants were subproducts of matrix turnover rather than newly synthesized. To our knowledge, this study is the first one so far applying SILAC technology in the context of cartilage and chondrocytes physiology.