The inverse electron demand Diels-Alder conjugation reaction has gained increasing importance over the past few years for efficient in vivo and ex vivo radiometal labeling of antibodies. However, the application of this very fast reaction type has not been studied for radiolabeling of peptides so far. We show here the synthesis of 3-benzyl-1,2,4,5-tetrazine-comprising ((1,4,7,10-tetraazacyclododecane-4,7,10-triyl)triacetic acid-1-glutaric acid) (DOTA-GA) and ((1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) (NODA-GA) chelators and their radiometal labeling with 68Ga3+ and 64Cu2+. The secondary labeling precursors 68Ga-DOTA-GA-Tz, 68Ga-NODA-GA-Tz, and 64Cu-DOTA-GA-Tz were obtained in high radiochemical yields (RCYs) and purities as well as molar activities for further labeling of trans-cyclooctene (TCO)-modified peptides. However, the following reactions of the radiometal-labeled tetrazines with different TCO-comprising model peptide analogs unexpectedly resulted in the formation of a considerable amount of side products (20-55%) which limits the overall achievable RCYs and purities as well as molar activities of the target radiopeptides. Under otherwise identical, nonradioactive reaction conditions, this effect could however not be observed. In contrast, the corresponding one-step radiolabeling protocols provided the target 68Ga-labeled radiopeptides in exceptionally high RCYs and purities of ≥99% and molar activities of 68-72 GBq/μmol starting from activities of 340-358 MBq of 68Ga. Thus, the usefulness of the two-step labeling of TCO-modified peptides with radiometal-labeled chelator-tetrazines seems to be limited.