Protoplasts isolated from hypocotyl tissue of dark-grown soybean ( Glycine max L.) seedlings, were labelled with fluorescein, using fluorescein diacetate, and fused electrically with green mesophyll protoplasts from seedling cotyledons of a wild perennial relative. The resulting heterokaryons were bifluorescent owing to the green fluorescent label of the soybean parent and the red chlorophyll autofluorescence from the cotyledon protoplasts of the wild Glycine sp. G1171. Post-fusion protoplast populations comprised between 1.61% and 7.40% heterokaryons with a mean of 3.77 ± 2.13%. The heterokaryons were isolated from unfused protoplasts and homokaryons by flow cytometry. They were cultured in agarose-solidified medium, where they developed into cell colonies. The latter produced friable callus if transferred to a rich culture medium, or green, nodular tissue and occasionally abnormal shoots, if grown on regeneration media previously used for Glycine species. Phenotypically normal shoots were obtained when callus on SC2 medium was transferred to SC2 supplemented with kinetin, zeatin and gibberellic acid (GA 3). However, these shoots did not develop further. Polyacrylamide gel electrophoresis of total protein revealed that heterokaryon-derived tissues grown on both K8 and SC2 media produced more isoenzymes of aspartate aminotransferase than an exact summation of the parental zymograms.