A variety of peptide ligands are known to inhibit the function of neuronal nicotinic acetylcholine receptors (nAChRs), including small toxins and brain-derived peptides such as beta-amyloid(1-42) and synthetic apolipoproteinE peptides. The myristoylated alanine-rich C kinase substrate (MARCKS) protein is a major substrate of protein kinase C and is highly expressed in the developing and adult brain. The ability of a 25-amino acid synthetic MARCKS peptide, derived from the effector domain (ED), to modulate nAChR activity was tested. To determine the effects of the MARCKS ED peptide on nAChR function, receptors were expressed in Xenopus laevis oocytes, and two-electrode voltage-clamp experiments were performed. The MARCKS ED peptide completely inhibited acetylcholine (ACh)-evoked responses from alpha7 nAChRs in a dose-dependent manner, yielding an IC(50) value of 16 nM. Inhibition of ACh-induced responses was both activity- and voltage-independent. The MARCKS ED peptide was unable to block alpha-bungarotoxin binding. A MARCKS ED peptide in which four serine residues were replaced with aspartate residues was unable to inhibit alpha7 nAChR-mediated currents. The MARCKS ED peptide inhibited ACh-induced alpha4beta2 and alpha2beta2 responses, although with decreased potency. The effects of the MARCKS ED peptide on native nAChRs were tested using acutely isolated rat hippocampal slices. In hippocampal interneurons, the MARCKS ED peptide was able to block native alpha7 nAChRs in a dose-dependent manner. The MARCKS ED peptide represents a novel antagonist of neuronal nAChRs that has considerable utility as a research tool.