Objectives: In-vitro investigation of human pulp-derived cells is hampered by the fact that primary cells in culture undergo senescence and thus have a limited lifespan. However, senescence can be overcome by transfection of primary cells with oncogenes like SV40. The objective of this work was to establish immortalized cells maintaining phenotypic characteristics of odontoblast-like cells. Methods: Passage 3 primary human pulp cells were transfected with a plasmid containing coding sequences of SV40ltAg. Transformed human pulp cells (t-HPC) were established and integration of SV40 was evaluated both by PCR and histochemistry. Immortalization was investigated by comparing the potential of primary and transfected cells of growing to confluency after subsequent passaging. For determination of cell type vimentin and cytokeratin were detected by histochemistry. Gene expression of collagen I and III, alkaline phosphatase, osteocalcin, bone sialoprotein, dentin sialophosphoprotein and dentin matrix protein I was evaluated by RT-PCR in primary cells passage 3 and transfected cells passages 10 and 25. Results: A cellular crisis appeared in passage 2 after transfection, whereupon several clones could be established. Stable integration of SV40ltAg sequences into the pulp cells genome was verified by PCR as well as by histochemistry. Primary human pulp cells were cultured until senescence up to passage 7, transfected cells showed an extension of life span and were cultured to passage 20 after transfection. At this point a second crisis appeared, after which proliferation started anew and one immortalized clone could be established. Both primary and transfected cells were identified as fibroblasts showing signals to histochemical staining for vimentin and no signal to cytokeratin. RT-PCR revealed tissue-specific gene expression of the above mentioned marker proteins. Conclusion: SV40 could successfully be integrated in human pulp-derived cells and led to immortalization. Transfected cells appear alike primary cells and can be characterized as odontoblast-like cells.