Resistance to the non-selective herbicide dl-phosphinothricin (PPT) was introduced into commercial Lotus corniculatus cv. Bokor by co-cultivation of cotyledons with Agrobacterium tumefaciens AGL1 harbouring the binary vector pDM805 which contains the bialaphos resistance gene (bar) from Streptomyces hygroscopicus encoding phosphinothricin acetyltransferase (PAT) and the uidA gene encoding β-glucuronidase. The half-cotyledon explants were precultured on regeneration Murashige and Skoog's (MS) medium supplemented with 6-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA) at 0.5 mg L−1 each, 3 days prior to infection. Upon co-cultivation, the explants were cultured on PPT-free regeneration medium for 10 days, and then subcultured on regeneration/selection media with increasing PPT concentrations (5–7 mg L−1) for about 18 weeks. Out of 480 initially co-cultivated explants, 272 regenerated shoots survived the entire PPT selection procedure. Resistant shoots were grown further, multiplied by tillering that was additionally promoted by PPT and rooted on hormone-free MS medium containing 5 mg L−1 PPT. Established shoot cultures, continuously maintained on the same medium, have preserved PPT resistance up to now (more than 2 years). Transformed plants assessed in vitro and in a greenhouse were tolerant to the herbicide PPT at 300 mg L−1 equivalent to more than twofold the recommended field dosage for weed eradication. Applied PPT treatment did not affect the activities of glutamine synthetase (GS; EC 220.127.116.11) and NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 18.104.22.168) in transformed plants. However, PPT did increase the mobility of glutamine synthetase isoforms GS1 and GS2 as well as the inhibition of an additional high mobility GS (hmGS) activity. In untransformed plants, PPT treatment reduced total GS activity by 4.4-fold while contrary the activity of NADH-GDH was increased by ninefold. All transformed herbicide-resistant plants were phenotypically normal and exhibited genomic stability, as were the untransformed plants analysed by flow cytometry. Under greenhouse conditions, they grew to maturity, flowered and set seeds. Stable integration and expression of the bar gene in T0 and T1 plants were confirmed by Southern and Western blot analysis, while integration of the reporter uidA gene did not occur. The bar gene was inherited in a Mendelian fashion by the progeny, as detected by PPT resistance. The production of PPT-resistant plants may have significant practical applications in weed control in fields of L. corniculatus.