Background Screening libraries against a molecular target in vitro are idealized models that cannot reflect the real state in vivo where biomolecules coexist and interact. C-terminal amide tripeptides labelled with Technetium-99m can provide a unique noninvasive approach to trace a large number of compounds in vivo.Methods The C-terminal amide tripeptide libraries were synthesized on Rink Amide-MBHA resin using iterative and pooling protocol. Technetium (Ⅴ) oxo core [TcO3+] was bound to each tripeptide via 4 deprotonated nitrogen atoms to form a library of 8000 99mTc tripeptoid complexes. The radiocombinatorial screening (RCS) in vivo was carried out on SD rats and A549 tumour bearing mice.Results Signals of tissue distribution and metabolism of libraries were recorded by counting or imaging and tissue targeting leads identified by both random and directed RCS. Among them, 99mTc RPA, 99mTc VIG and 99mTc RES had specific tissue targeting in kidney, liver and tumour respectively. The percent injected dose per gram tissue of 99mTc labelled leads in their target tissue was highly structure dependent. Because the nontarget tissue binding and the metabolism of 99mTc tripeptoid sublibraries were simultaneously monitored successfully by RCS, the interference of background activity was limited to the lowest level. Optimization of renal function agent from the labelled libraries was carried out by directed screening. 99mTc DSG was finally identified the most promising agent for renal function studies.Conclusions RCS in vivo is a powerful tool for the discovery of tissue targeting drugs. The potential screening bias is probably the major limitation of labelled libraries.