The immature nucleus pulposus (NP) is populated by cells of notochordal-origin that are larger and contain an extensive cytoskeletal network and numerous vacuoles. The disappearance of these cells with age is believed important in regulating metabolic shifts that may contribute to age-related disc degeneration. The precise biological function of these notochordal cells in the immature NP remains unclear, however, because of challenges in studying the mixed cell population in the NP. In this study, notochordal-like cells were purified from immature NP cells using a new fluorescence-activated cell sorting (FACS) protocol with auto-fluorescence and size analysis. The unique molecular phenotypes of sorted notochordal-like cells were characterized by the mRNA expression pattern for key matrix proteins and modulators, and by the expression of cell–matrix receptor integrin subunits. An FACS analysis showed that the immature NP contained a majority of cells that were larger than anulus fibrosus (AF) cells and with fluorescence higher than AF cells. In comparison with the small NP cells separated by the FACS protocol, sorted notochordal-like cells expressed lower mRNA levels of type I collagen, biglycan, TIMP1, HSP70 and c-fos, and did not express detectable mRNA levels of decorin, lumican, multiple MMPs or IL-1β via real-time quantitative RT-PCR. A greater number of these notochordal-like cells also expressed the higher levels of α6, α1 and β1 integrin subunits as compared to small NP cells. Together, our results point towards a unique molecular phenotype for these notochordal-like cells of NP, characterized by the absence of gene expression for specific small proteoglycans and higher protein expression of integrin subunits that regulate interactions with collagens and laminin. Future studies will be important for revealing if this unique molecular profile is coordinated with functional differences in pericellular matrix regions and/or integrin-mediated cell–matrix interactions for these notochordal-like cells within the NP.