The purpose of this study was to evaluate different protocols of enzymatic treatment (collagenase with either protease, trypsin or hyaluronidase) to isolate mature odontoblasts. Primary odontoblasts were obtained from human molars, which was confirmed by histology and scanning electron microscopy. The combination of collagenase with protease appeared most suitable and resulted in higher cell numbers and better integrity of the odontoblast processes, whereas combination with hyaluronidase or trypsin led to truncated processes and detachment of cell patches instead of single cells. However, trypan blue staining after 24 h showed that odontoblasts in culture did not remain viable. Gene expression analysis was possible after mRNA extraction from tissues ex vivo and real-time semi-quantitative PCR revealed increased expression of collagen, nestin, bone sialoprotein and dentin matrix acidic phosphoprotein 1 in the odontoblast layer. Though primary odontoblasts could not be cultivated after isolation, characteristic genes were identified to differentiate odontoblasts from pulp fibroblasts.