This paper reports a study of rhodopsin's structure and function using insertional mutagenesis with a flexible epitope. Sixteen rhodopsin derivatives were constructed, each of which carried a 12-amino acid epitope derived from the c-Myc protein flanked by penta-glycine linkers. For eight of the insertion mutants, the membrane sideness of the epitope insert was determined by immunostaining of intact or permeabilized cells. The results confirm the sidedness of each of the six helix connecting loops and the amino and carboxyl termini as postulated by the current seven-helix models of G-protein-coupled receptors and provide the first experimental evidence for the existence of the third extracellular loop. In general, inserts that were either closer to the amino terminus or on the extracellular face were more likely to disrupt folding and/or stability than were inserts near the carboxyl terminus or on the cytosolic face. Epitope insertion at positions 139 or 239, in the second and third cytosolic loops, respectively, failed to activate transducin, whereas an insertion at position 333 in the carboxyl-terminal tail was fully functional. The experimental approach described here should prove generally useful for elucidating structural and functional properties of both membrane and globular proteins.