When agitated, Atlantic hagfish (Myxine glutinosa) produce large quantities of slime that consists of hydrated bundles of protein filaments and membrane-bound mucin vesicles from numerous slime glands. When the slime exudate contacts seawater, the thread bundles unravel and the mucin vesicles swell and rupture. Little is known about the mechanisms of vesicle rupture in seawater and stabilization within the gland, although it is believed that the vesicle membrane is permeable to most ions except polyvalent anions. We hypothesized that the most abundant compounds within the slime gland exudate have a stabilizing effect on the mucin vesicles. To test this hypothesis, we measured the chemical composition of the fluid component of hagfish slime exudate and conducted functional assays with these solutes to test their ability to keep the vesicles in a condensed state. We found K+ concentrations that were elevated relative to plasma, and Na+, Cl− and Ca2+ concentrations that were considerably lower. Our analysis also revealed high levels of methylamines such as trimethylamine oxide (TMAO), betaine and dimethylglycine, which had a combined concentration of 388 mmol l−1 in the glandular fluid. In vitro rupture assays demonstrated that both TMAO and betaine had a significant effect on rupture, but neither was capable of completely abolishing mucin swelling and rupture, even at high concentrations. This suggests that some other mechanism such as the chemical microenvironment within gland mucous cells, or hydrostatic pressure is responsible for stabilization of the vesicles within the gland.