Matrix metalloproteinases (MMPs) play an important role in tooth development and influence caries development and hybrid layer degradation. Literature is scanty on the differences in the activity of MMPs between primary and permanent dentine. Accordingly, the aim of the present study was to investigate endogenous gelatinolytic activity in primary and permanent dentine. Separate batches of dentine powder were obtained from intact human primary and permanent molars (n=6). Each batch was divided in two subgroups: (1) mineralized; (2) demineralized with 10% H3PO4. After protein extraction, gelatine zymography was performed. Further, in-situ zy-mography was done on dentine sections of the same groups (n=3). The slices were polished, cov-ered with fluorescein-conjugated gelatine and evaluated using a confocal microscope. In-situ zy-mography data were analysed using two-way analysis of variance and post-hoc Holm-Sidak statistics (α=0.05). Primary dentine showed poorly-defined bands in the zymograms that vaguely corresponded to the pro-form and active form of MMP-2 and the pro-form of MMP-9. In perma-nent dentine, demineralized powder demonstrated stronger gelatinolytic activity than mineral-ized powder. In-situ zymography identified stronger enzymatic activity in primary etched den-tine (p<0.05). Stronger enzymatic activity recorded in primary dentine may be related to the dif-ferences in morphology and composition between primary and permanent dentine.