Mutations in myocilin (MYOC) associate with glaucoma and ocular hypertension. Unfortunately, the specific role of MYOC, a widely expressed protein of unknown function, in ocular hypertension is unknown. Since MYOC localizes both to intracellular membranes and to the cytosol, we tested the hypothesis that MYOC is a cytosolic protein that associates with cellular membranes via its coiled-coil domain. Using green fluorescent protein (GFP) chimeras in expression and metabolic labeling studies, we observed that MYOC's putative signal peptide failed to traffic GFP into the secretory machinery and out of transfected cells. Next, we tested which of MYOC's three folding domains were responsible for targeting. In cell fractionation and immunofluorescence microscopy studies, the coiled-coil, but not the helix-turn-helix or olfactomedin domains, was necessary and sufficient to target GFP chimeras to cell membranes. Interestingly, a vesicular phenotype required sequential addition of the helix-turn-helix and olfactomedin domains to the coiled-coil. Taken together, these data indicate that the coiled-coil domain, not the putative signal sequence, is responsible for the targeting of MYOC to the secretory machinery.