Assessment for the potential of liver toxicity during drug development currently utilises primary hepatocytes and animal models; however, these are costly and present ethical issues. Secondary heptocytes, derived from hepatic carcinomas, do not retain all the features of hepatocytes in vivo such as expression of key metabolic enzymes and are of limited value. To offer an alternative to current models, this research has attempted to differentiate the secondary hepatic cell lines HepG2 and Huh7 to produce cells which more closely resemble the in vivo hepatocyte. Initially, adaptations of protocols for differentiation of stem cells into hepatocyte-like cells (1-4) were used; combinations of hepatocyte growth factor (HGF), fibroblast growth factor 4 (FGF4), and oncostatin M (OSM) along with dexamethasone, insulin-transferrin-selenium (ITS) and sodium pyruvate. Treatment times were limited to 4 days due to overgrowth and death of cells. To restrict growth, 1% di-methyl-sulfoxide (DMSO) was used (5) and cells were treated for up to 27 days. Finally, DMSO was combined with a selection of growth factors and applied for up to 25 days. RNA was extracted at regular intervals throughout the treatments, reverse transcribed, and abundance of specific mRNAs determined by real-time PCR. mRNA levels of genes associated with the differentiated hepatic phenotype such as albumin and transferrin, more abundant in mature than immature hepatocytes, were the focus of initial analysis. When treated with growth factors only, cells showed no clear indication of maturation, possibly due to the relatively short incubation time. Increased levels of albumin mRNA were seen with 1% DMSO in both HepG2 and Huh7 cells, following 15 and 9 days treatment respectively (HepG2, control = 133.1±26.2 (mean±S.E.M.), 15 days DMSO = 516.9±74.7, p<0.01. Huh7, control = 123.8±14.2, 9 days = 302.7±64.4, p<0.01 tested by 1-way ANOVA with Dunnett’s post test, n=3. All data is given in arbitrary units.). HepG2 cells also showed significant increases in albumin and transferrin mRNAs after 5 to 10 days of treatment with HGF and 1% DMSO (albumin control = 133.1±26.2, 5 days = 308.2±38.8, 10 days = 686.0±61.1, p<0.01 for both. Transferrin control = 0.43±0.13, 5 days = 1.08±0.20, 10 days = 1.76±0.22, p<0.01 for both, n=6). Huh7 cells showed no signs of increased differentiation during growth factor plus DMSO treatment. These results indicate that with further refinement of treatment, a secondary hepatocyte may be altered to show a more differentiated hepatocyte phenotype.