Recent evidence suggests that agricultural and horticultural crops may be able to take up significant quantities of dissolved organic nitrogen (DON). Our aims were to determine the effects of supplying different forms of N on growth and on the activities of N-assimilatory enzymes in tomato. Two genotypes of tomato were grown in sterile hydroponic culture without N (control), with NO3– or NH4+ (3 mM), or with organic-N in the form of glycine (1.5, 3.0, or 6.0 mM). The results showed that biomass production and N-contents were similar in both genotypes when supplied with NO3– or with glycine, and that this growth was much greater than in plants supplied with NH4+ alone, or without added N. In addition, the production of plant biomass was positively correlated with the concentration of glycine-N used; however, the magnitude of the response was genotype-dependent. The form of N supplied also significantly affected the activities of several key N-assimilatory enzymes in roots and shoots. For example, addition of glycine increased the activities of NADH-glutamate dehydrogenase (NADH-GDH), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) in roots, compared with the NO3– or NH4+ treatments. Our results clearly demonstrate the intrinsic ability of tomato plants to use DON as a sole source of N. Further studies to investigate the functional significance of DON in horticultural systems under non-sterile conditions are therefore warranted.