Summary An improved procedure has been developed for the fusion of plant protoplasts based on the use of polyethylene glycol possessing a low carbonyl content. Different commercial preparations of PEG were analysed using a sensitive carbonyl assay procedure and only those with a very low carbonyl content (< 5 × 10 −4M) were used. Sterilisation by membrane filtration was employed, since autoclaving markedly increased the carbonyl content with associated deleterious effects on fusion frequency and heterokaryon viability. Elution of PEG was carried out using a hypotonic mannitol solution at pH 5.8, without added Ca 2+, immediately followed by 9 % w/v mannitol (pH 5.8), again without added Ca 2+. This improved PEG fusion procedure has been found to be applicable to a range of protoplast combinations. Its use results in a high fusion frequency with good resultant heterokaryon viability and a low level of clumping. Comparisons indicate that it is a satisfactory alternative to electrofusion.