The vitelliform macular dystrophy type 2 (VMD2) gene mutated in Best macular dystrophy encodes a 585-amino acid putative transmembrane protein termed bestrophin-1. The vast majority of known disease-associated alterations are of the missense type, which cluster near predicted transmembrane domains (TMDs). To investigate bestrophin-1 membrane topology and to assess consequences of point mutations on membrane integration, we have analyzed the insertion of putative TMDs into the endoplasmic reticulum (ER) membrane. Out of six potential TMDs, our data suggest a topological model of bestrophin-1 with four transmembrane-spanning segments and one large cytoplasmatic loop between putative TMD2 and TMD5. Consequently, a relatively hydrophobic segment containing putative TMD3 (aa 130-149) and TMD4 (aa 179-201) is located within the cytoplasm. Furthermore, we show that three out of 18 disease-associated alterations investigated (I73N, Y85H, F281del) reveal measurable effects on membrane insertion suggesting that defective membrane integration of bestrophin-1 may represent a potential disease mechanism for a small subset of Best macular dystrophy-related mutations.