In ovarian follicles of Drosophila, changes in external osmolarity affect the steady-state potentials of oocytes more than those of nurse cells. Thus the osmolarity of the incubation medium affects the occurrence and the direction of an electrical gradient across the connecting intercellular bridges. At 255 mOsm nurse cell Em averaged 2.5 mV negative to oocyte Em (P < 0.001). At 275 and at 300 mOsm there was no significant difference between oocyte and nurse cell. At 400 mOsm nurse cell Em averaged 1.1 mV positive to oocyte Em (P = 0.007). The osmolarity of adult Drosophila hemolymph was measured by a variation of freezing point depression and averaged 251 +/- 9 (SE) mOsm. The measured osmolarity and measured ionic concentrations of adult Drosophila hemolymph were used to develop an incubation medium, which was used to incubate developing ovarian follicles. Electrical measurements made in this saline, which mimics in vivo conditions, confirmed reports of a nurse cell-oocyte electrical gradient, with nurse cell Em significantly more negative than oocyte Em. Microinjections of the negatively charged dye Lucifer yellow CH showed that this charged molecule accumulated in the oocyte at the in vivo osmolarity, and in the nurse cells at highly elevated osmotic levels.