For many studies, transient transfections are of limited use due to a low percentage of cells transfected and short-term duration of gene expression, whereas construction of stable cell-lines can be labour-intensive and take many months. We report the use of an episomal replicating vector and characterise its use in the simple and rapid production of stable cell lines using a fluorescent reporter protein in two cell lines. The cDNA for a variant of enhanced yellow fluoresecent protein (EYFP; Jayaraman et al. 2000) was sub-cloned into the SalI restriction sites of pCEP4 (Invitrogen Ltd). CFTE cells (Gruenert et al. 2004) were seeded at a density of 200,000 per 35 mm dish 24 h prior to transfection. 2μg DNA was mixed with 6μl Lipofectamine (Invitrogen Ltd) in 3 ml serum-free culture medium, incubated at room temperature for 15 min and added to cells. Medium was replaced after 5 h with fresh medium supplemented with 10% foetal calf serum. 24 h post-transfection, cells were observed by fluorescence microscopy and approximately 10% of cells were expressing EYFP. Cells were treated with hygromycin to kill non-transfected cells. Over a 21 day period, the percentage of cells expressing EYFP increased such that essentially all cells were expressing EYFP. When hygromycin was removed, essentially all cells remained yellow for a further 3 weeks, although a slight decrease in intensity was observed. SHSY-5Y cells (Vinores et al. 1984) were cultured and transfected in a similar manner. 24 h post-transfection approximately 10% of cells were judged to be expressing the variant EYFP. Over a 21 day period, in the absence of hygromycin, the percentage of cells expressing variant EYFP remained constant. Towards the end of this period, a few colonies comprising a small number (less than 10) of yellow cells began to emerge within the population. All cells in each colony had the same level of fluoresence, but the level of fluorescence differed between colonies. Such an observation is consistent with the integration of the pCEP4-EYFP plasmid into different regions of the genome resulting in different levels of gene expression. Colonies such as this would be expected to give rise to conventional stable cell lines. However, to ensure a homogeneous stable population, they would first need to be cloned, and then amplified. This would take at least another 3 weeks to reach 10⁶ cells. In summary, the use of episomally replicating plasmids, such as pCEP4, can be used to generate stably expressing cells in the absence of hygromycin selection, or select stable cell-lines within 3 weeks in the presence of hygromycin selection, with minimal manipulations. When hygromycin selection is withdrawn, gene expression remains stable for at least 3 weeks.