We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum 1.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Clucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, 8-glucuronidase (GUS), neomycin phosphotransferase, and an a-amylase inhibitor were used to transform subterranean clover. lransgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of 1-DNA into the subterranean clover genome. l h e expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding CUS segregated as a dominant Mendelian trait. ~ Subterranean clover (Trifolium subterraneum L., subclover) is native to the Mediterranean regions and is grown as a pasture legume mainly in Australia and the countries around the Mediterranean. To a lesser extent, it is also grown in the Americas, Africa, New Zealand, and Japan (Johnstone and McLean, 1987). In Australia, subterranean clover is the major pasture legume and is grown on more than 16 million ha of mainly acidic and infertile lands. As a pasture crop and because of its ability to fix nitrogen, subterranean clover is a major contributor to the productivity of the meat, wool, dairy, and wheat industries in Australia (Johnstone and McLean, 1987). It also has the potential to be grown in other temperate regions of the world. A number of grain and pasture legumes are now amenable to gene transfer by genetic engineering using the Agrobacte-rium-mediated gene delivery system. These include soybean (Hinchee et al.