Enamel and dentin at the cervical portion of the tooth are frequently covered by a collagen-free matrix referred to as acellular afibrillar cementum (AAC). It is believed that AAC deposition occurs when the enamel organ is displaced or disrupted, and mesenchymal cells from the dental follicle gain access to the tooth surface, differentiate into cementoblasts, and secrete noncollagenous proteins typically found in collagen-based mineralized tissues. A similar thin layer of mineralized matrix is found at the enamel-free area (EFA) of rodent molars, but in this case the matrix is covered by inner enamel epithelium (IEE) throughout development. We have, therefore, used this site as a paradigm to test the hypothesis that typical mesenchymal matrix proteins can also be found in association with epithelial cells. To this end, we have analyzed the presence and distribution of enamel- and cementum-related matrix proteins at the EFA and at the cervical portion of the tooth. Rat mandibular molars were processed for colloidal gold immunolabeling with antibodies to amelogenins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and dentin sialoprotein (DSP), and the plasma proteins alpha 2 HS-glycoprotein and albumin. The EFA matrix was immunoreactive for amelogenins as well as for BSP, OPN, OC, and alpha 2 HS-glycoprotein, but not for albumin and DSP. The AAC was, similar to the EFA matrix, labeled for BSP, OPN, OC, and alpha 2 HS-glycoprotein. These data show for the first time that the EFA matrix is comprised of a mixture of enamel- and cementum-related proteins, a situation that parallels the distribution of matrix constituents at the cervical portion of the tooth. Since the EFA matrix is deposited on top of the mineralized dentin, and since the enamel organ seals off the forming matrix, it is concluded that EFA cells are responsible for the production of these proteins. Consistent with previous reports showing that epithelial cells can produce both BSP and OPN in some circumstances, the data also suggest that AAC may be deposited by cells of epithelial origin. Furthermore, they lend support to the possibility that cells derived from Hertwig's epithelial root sheath may likewise be capable of producing cementum matrix proteins.