It has been reported that unlike the more commonly expressed splice variants, the embryonic and stress-associated readthrough form of acetylcholinesterase (AChE-R) is unable to promote cell adhesion and neurite outgrowth. We investigated the possibility that the unique AChE-R C-terminal peptide (ARP) might be responsible for this difference, either by binding to AChE itself and inactivating the adhesion-mediating site or by competing with AChE for ligand binding. Synthetic peptides representing the ARP, a scrambled version of the ARP, and sequences of the previously identified adhesion-mediating site on AChE were used in in vitro binding and neuroblastoma cell-spreading assays. It was observed that the ARP was able to bind to laminin-1, identified previously as an in vitro AChE ligand and, to a lesser extent, to collagen IV and to AChE itself. ARP-AChE binding was, however, of very low affinity and was not significantly affected by peripheral site inhibitors, suggesting that inactivation of the AChE adhesion site is not the reason for AChE-R's antiadhesive character. On the other hand, the ARP competed with AChE and the adhesion site peptides for binding to laminin in vitro, and the ARP was observed to inhibit cell spreading in neuroblastoma cells grown on laminin. Monoclonal antibodies recognizing the known AChE adhesion site reacted with the ARP, suggesting structural similarities. These were borne out by an examination of sequence alignments of the ARP and the 28-53 AChE sequence. The ARP contains part of the PPxxxxRFxPPEP motif seen in AChEs and cholinesterase-domain proteins, and both it and the 37-53 sequence bear some resemblance to collagen and collagen-like proteins. It therefore appears likely that the ARP's structural similarity to the AChE adhesion-mediating site is the basis for the observed competition for ligand binding and might account for the antiadhesive characteristics of AChE-R.