The effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and the possible mechanisms were investigated in this study. 3T3-L1 preadipocytes were cultured in vitro and treated with different concentrations of ghrelin. Proliferation of 3T3-L1 preadipocytes was evaluated by MTT method and mRNA levels of c-myc and thymidine kinase were detected by RT-PCR. Morphological changes of 3T3-L1 preadipocytes were observed and cell differentiation was measured by oil red O staining. The mRNA levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and CAAT/enhancer binding protein (C/EBPalpha) in the cells at different differentiation stages were detected by RT-PCR. The results showed that ghrelin at concentrations of 10(-7) to 10(-15) mol/L could significantly promote preadipocyte proliferation (P<0.05), with the most pronounced effect observed at 10(-11) mol/L (P<0.01). Treatment of 3T3-L1 preadipocytes with ghrelin significantly increased the mRNA levels of c-myc and thymidine kinase (P<0.01). Morphological findings demonstrated that the great amount of lipid droplets appeared in the 3T3-L1 preadipocytes treated with ghrelin. Ghrelin could morphologically induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Ghrelin significantly increased the mRNA levels of PPARgamma and C/EBPalpha during the differentiation, when compared with control group (P<0.05). The mRNA levels of PPARgamma and C/EBPalpha were obviously up-regulated with the differentiation of preadipocytes after the treatment of ghrelin. There were significant difference in the mRNA levels of PPARgamma and C/EBPalpha on day 2 and day 8 of the differentiation of 3T3-L1 preadipocytes (P<0.01). In conclusion, ghrelin could promote the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARgamma and C/EBPalpha and therefore enhance the sensitivity of adipocytes against insulin.