Humanin (HN) and HN-derivatives are a family of peptides first reported in the last decade with potent in vitro and in vivo neuroprotective activity, which is mediated through a not completely elucidated mechanism. Recently, our group has evaluated the effect of various HN-derivatives on the 3-quinuclidinyl benzilate (QNB)-induced impairment of spatial orientation and memory in rats, by employing the T-maze test. In the present work four new, tyrosine containing HN-derivatives were synthesized (Y-PAGASRLLLTGEIDLP, peptide I; Y-PAGASRLLLLTGEIDLP, peptide II; Y-SALLRSIPAPAGASRLLLTGEIDLP, peptide III; Y-SALLRSIPAPAGASRLLLLTGEIDLP, peptide IV). The neuroprotective action of these peptides was evaluated in the T-maze test and the most active among them (peptides I and III) was radiolabeled with (125)I. The pure monoradioiodinated peptides were used in: (i) in vitro binding studies with various neuronal cell lines and with brain and stomach membranes from rats and mice and (ii) in vivo biodistribution studies in rats and mice. Moreover, the metabolic stability of the above radiolabeled peptides was studied. Under the experimental conditions used, our data do not confirm the existence of specific binding sites for HN on the neuronal tissue. Nevertheless, they are setting the basis for further relevant studies aiming at the clarification of the mode of the neuroprotective action of HN-peptides.