Relatively few studies comparing different methods of labelling peptides with 99Tcm have been reported. In this investigation, we evaluated the influence of three chelators on the in vitro and in vivo properties of two small, similar peptides (HNE2 and HNE4) labelled with 99Tcm. Both peptides were labelled with hydrazinonicotinamide (HYNIC) (tricine) at pH 5-6 and with diethylenetriaminepentaacetic acid (DTPA) and mercaptoacetyltriglycine (MAG3) at both pH 5-6 and 7-8. All ten preparations were brought to pH 7.2 immediately after labelling. Each preparation labelled well and control labelling showed each label to be attached specifically at chelation sites. Analysis of 37 degrees C human serum incubates showed little evidence of label instability but high protein binding in several cases. The stability of 99Tcm to cysteine challenge for labelled DTPA- and MAG3-peptides was similar but lower than that for the HYNIC-peptides. Reverse phase HPLC of the DTPA-peptides, but not the MAG3-peptides, showed different 99Tcm species depending on labelling pH. The 3 h biodistributions in normal mice were generally independent of labelling pH for both MAG3-peptides but were heavily influenced by labelling pH for both DTPA-peptides. While significant differences in biodistribution for the same labelling method were evident between peptides, as expected, far larger differences in the case of both peptides resulted from changing chelators and, in the case of DTPA, changing the labelling method. In summary, the chelators and labelling methods influenced the biodistribution of 99Tcm in a characteristic fashion common to both peptides. Differences in biodistribution due to the different peptides were relatively small and generally lost in the much larger differences due to chelator and labelling method. In conclusion, it may be important to compare chelators and labelling methods before selecting a 99Tcm labelling method for any particular peptide.