Transporters for di-and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.