The transmembrane disposition of the visual pigment rhodopsin was studied by the covalent labeling of protein amino groups with membrane-permeable and -impermeable imidoesters. A new, highly reactive permeable reagent, 2-(methylsulfonyl)ethyl acetimidate (SAI) was developed for this purpose. The permeabilities of both this compound and the "impermeable" reagent isethionyl acetimidate (IAI) across the rod outer segment disk membrane were directly measured. Our results indicate that rhodopsin contains three classes of amino groups. One class (35--55% of the total) reacts rapidly with the membrane-impermeable reagent and is presumably exposed on the outside surface of the membrane. A second class (35--55% of the total) is located on the internal surface of the disk since its rate of reaction is dependent on the relative permeabilities of the labeling reagents. The remaining 10% of the rhodopsin amino groups are inaccessible to either type of imidate and are largely accounted for by the single lysine residue which specifically binds the chromophore retinal. These findings, taken together with evidence from freeze--fracture electron microscopy, imply that rhodopsin is a transmembrane protein.