MARCKS is postulated to regulate the passage of secretory granules through cortical actin in the early phase of exocytosis. There are, however, three proposed mechanisms of action, all of which were derived from studies using synthetic peptides representing either the central phosphorylation site domain, or the upstream, N-terminal domain: it tethers actin to the plasma membrane and/or to secretory granules, and/or it sequesters PIP₂. Using MARCKS null mice, we probed for a loss of function secretory phenotype in mast cells harvested from embryonic livers and maturated in vivo (eHMCs). Both WT and MARCKS null eHMCs exhibited full exocytic responses upon FcRI receptor activation with DNP-BSA, whether they were in suspension or adherent. The secretory responses of MARCKS null eHMCs were consistently higher than that of WT cells, but the differences had sporadic statistical significance. The MARCKS null cells exhibited faster secretory kinetics, however, achieving the plateau phase of the response with a t_1/2 ~2.5-fold faster. Hence, MARCKS appears to be a non-essential regulatory protein in mast cell exocytosis, but exerts a negative modulation. Surprisingly, the MARCKS N-terminal peptide, MANS, which has been reported to inhibit mucin secretion from airway goblet cells (J Biol Chem 276: 40982-40990, 2001), inhibited hexosaminidase secretion from WT and MARCKS null eHMCs, leading us to re-examine its effects on mucin secretion. Results from studies using peptide inhibitors with human bronchial epithelial cells and with binding assays using purified mucins, suggested that MANS inhibited the mucin binding assay, rather than the secretory response.