Different gene transfer approaches to achieve long-term transgene expression in cultured primary bovine chondrocytes were compared using enhanced green fluorescent protein (EGFP) as a reporter. Transduction with a high-capacity adenoviral vector was 82% efficient when analysed by fluorescence microscopy, while up to 42% of plasmid-transfected cells were EGFP positive with FuGene as a transfection reagent. Rapid dominant marker selection of plasmid-transfected cells was achieved in monolayer culture. With either method of gene transfer, a high proportion of the chondrocytes remained transgene positive during prolonged alginate culture. Transgene transcription in single cells was quantified with a confocal laser scanning microscope. Detection of EGFP expression was more sensitive with this method, identifying more transgene-expressing cells than conventional fluorescence microscopy. Long-term EGFP expression was higher in adenovirally transduced chondrocytes embedded in alginate as compared to plasmid-transfected cells cultured in monolayer or in alginate. Both the adenoviral and the plasmid-based approach appear suited for studies of the molecular and cellular mechanisms by which mutations in cartilage matrix proteins cause disease.