The affinity toward the PAC1 receptor, the biological activity, and the alpha-helical content of several truncated PACAP27 analogs were measured. We first evaluated the pharmacological and structural parameters of C-terminal shortened PACAP fragments, from PACAP(1-23) to PACAP(1-19). All carboxy-truncated derivatives demonstrated circular dichroism spectra typical of a helical conformation. On the other hand, progressive shortening of the C-terminal domain gradually decreases the potency of PACAP to bind and to activate the PAC1 receptor. This decrease in biological activity was mainly attributed to the removal of residues that seem to interact directly with the receptor rather than to a destabilization of the C-terminal helical conformation. We also investigated the pharmacological and conformational characteristics of several hybrid PACAP27 derivatives containing an aliphatic molecular spacer connecting the N-terminal domain to the C-terminal region. However, this strategy revealed that none of these discontinuous analogs showed any significant affinity toward the PAC1 receptor, even if some of them exhibited circular dichroism spectra corresponding to an alpha-helical structure. This study suggests that several domains of PACAP27 are involved in the interaction with the PAC1 receptor and that the presence of the helical conformation is not a sufficient feature for receptor activation.