OBJECTIVE The purpose of this study was to investigate the effect of enamel matrix derivative on remineralisation of initial enamel carious lesions in vitro. METHODS Initial enamel carious lesions were created in bovine enamel blocks in vitro. The lesions were subjected to a pH-cycling regime of 24days. Each daily cycle included 4×3-min applications with one of four treatments: 1g/L NaF aqueous solution (positive control), 6% propylene glycol alginate (PGA) or distilled and deionised water (DDW) (both negative controls), and a gel of enamel matrix derivative and PGA (EMDgel). Samples were subjected to surface microhardness (SMH) testing, polarised light microscopy (PLM) and transverse microradiography (TMR) to measure SMH, mineral loss, lesion depth and mineral content of the surface layer and lesion body before and after pH-cycling. RESULTS NaF samples showed the highest SMH recovery of all the groups (P<0.05). EMDgel samples showed significantly higher SMH recovery than did PGA ones (P<0.05). NaF samples showed significantly less mineral loss and shallower lesions than all other groups (P<0.05). The DDW and EMDgel samples showed significantly less mineral loss and shallower lesions than PGA samples. Mineral deposition predominated much more at the surface layer in the EMDgel group than in the PGA group (P<0.05). CONCLUSIONS EMD, the active ingredient of EMDgel, may play an essential role in promoting remineralisation of initial enamel carious lesions. However, EMDgel as a whole did not cause detectable remineralisation of such lesions in vitro.