Rat intestinal fibroblast lines (F1:G9 and A1:F1) differing in their potential to support intestinal mucosal development were marked with reporter genes to investigate their differentiation potential. The fibroblasts were transfected with plasmids expressing either β-galactosidase (with or without a nuclear localisation signal) or green fluorescent protein (GFP). Transfection using Tfx50 or Fugene™ was more efficient than electroporation. The expression of β-galactosidase was more stable and stronger than GFP. Cells were optimally labelled using the plasmid pL27B-GAL, and sub-clones with a strong and uniform nuclear expression of β-galactosidase were isolated. These clones expressed β-galactosidase even after prolonged passage in the absence of selection. The β-galactosidase tagged lines (F1:G9galand A1:F1gal) retained the morphological characteristics, viability and differentiation properties of the parental non-transfected lines. In co-culture with a colorectal tumour cell line Caco-2, the F1:G9galand A1:F1galcells differed in their morphological organisation but this did not change their expression of smooth muscle α-actin.